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Full text of "Review of intramural research 1966"

R853 

.U5601 

1966 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 

INTRAMURAL 
RESEARCH 

1966 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 



ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 

Uhrwj 

(/. J. NATIONAL INSTITUTES OF HEALTH^^ 



Review 
of 

INTRAMURAL 
RESEARCH 

1966 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 
National Institutes of Health, Bethesda, Maryland 20014 



mo/ 



INSTITUTE RESEARCH DIRECTORS 



NATIONAL CANCER INSTITUTE 



NATIONAL HEART INSTITUTE 



NATIONAL INSTITUTE OF ALLERGY AND 
INFECTIOUS DISEASES 



Eugene J. Van Scott, M.D. 

Scientific Director for General 
Laboratories and Clinics 
C. Gordon Zubrod,M.D. 
Scientific Director for Chemotherapy 
Paul Kotin, M.D. 
Scientific Director for Etiology 
Nathaniel I. Berlin, M.D. 
Scientific Director for Clinical Research 

Robert W. Berliner, M.D. 

Scientific Director 
Donald S. Frederickson, M.D. 

Clinical Director 



John 



R. Seal, M.D. 
Scientific Director 
Vernon Knight, M.D. 

Clinical Director 

NATIONAL INSTITUTE OF ARTHRITIS AND 

METABOLIC DISEASES Joseph E. Rail, M.D., Ph.D. 

Scientific Director 
Robert S. Gordon, Jr., M.D. 

Clinical Director 

NATIONAL INSTITUTE OF CHILD HEALTH AND 

HUMAN DEVELOPMENT Roy Hertz, M.D. 

Scientific Director 

NATIONAL INSTITUTE OF 

DENTAL RESEARCH Seymour J. Kreshover, D.D.S., M.D., Ph.D. 

Scientific Director 
Edward J. Driscoll, D.D.S. 

Clinical Director 

NATIONAL INSTITUTE OF MENTAL HEALTH John C. Eberhart, Ph.D. 

Scientific Director 
Robert A. Cohen, M.D. 

Clinical Director 

NATIONAL INSTITUTE OF NEUROLOGICAL 

DISEASES AND BLINDNESS Karl Frank, Ph.D. 

Acting Scientific Director 
Maitland Baldwin, M.D. 

Clinical Director 

DIVISION OF BIOLOGICS STANDARDS Roderick Murray, M.D. 

Scientific Director 



G. Burroughs Mider, M.D. 
Director of Laboratories and Clinics 



William C. Mohler, M.D. 
Assistant to Director of Laboratories and Clinics 



111 



FOREWORD 

As the title implies this volume summarizes work done in the NIH 
laboratories and clinics and in the offices closely connected to these. It does 
not cover the research supported throughout the United States and in some 
other nations by grants from the various Institutes. Nor does it include 
research done on contract for the NIH unless this was a direct extension 
of an intramural program. 

This story covers the year from July 1965 through June 1966. The 
philosophy behind the Review remains the same as in previous years. 
These are the words of the staff who followed very general guidelines in 
writing their reports. They have received only minimal editing. Thus we 
have tried to preserve for the reader as direct a view as possible into our 
diverse and extremely active scientific community as its members pursue 
their investigations under the missions of the several Institutes and Divi- 
sions. Together these missions unite as the common purpose of the NIH, 
which strives to extend fundamental knowledge about the health and 
diseases of mankind. 



\^tf\Uo (S.SUiw 



James A. Shannon, M.D. 



CONTENTS 



Foreword 



NATIONAL CANCER INSTITUTE 

General Laboratories and Clinics 

Introduction 

The Laboratories 

The Clinical Branches 

Immunologic Research 

Macromolecular Biology Section 

Laboratory of Biochemistry 

Cytochemistry Section 

Nutrition and Carcinogenesis Section 

Tumor-Host Relations Section 

Nucleic Acids Section 

Protein Chemistry Section 

Laboratory of Biology 

The Thymus in Immunology 

Tumor Viruses 

Ultrastructure Cytology 

Plasma Cell Tumors 

Biochemical Genetics 

Genetics of Tumors 

Hepatic Carcinogenesis 

Malignant Transformation In Vitro 

Methodology in Tissue Culture 

Biochemistry 

Laboratory of Pathology 

Introduction 

Experimental Research 

Cancer in Man, and Related Animal Studies 

Cancer in Animals 

Induction and Pathogenesis 

Modifications in Carcinogenesis 

Biologic Factors in Neoplasia 

Spontaneous Tumors 

Transplanted Tumors 

Viral Carcinogenesis, and Problems of Immunology 

Polyoma Virus 

SV40 and Adenovirus 

Leukemogenic Viruses 

Virus Replication 

Interferon 



Page 
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8 
9 

11 
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26 
27 
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28 
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29 
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32 
34 
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37 



vii 



V"i CONTENTS 

Page 

Accumulation of Data on Laboratory Animals 37 

Phylogenetic Aspects of Neoplasia 38 

Development of New Techniques 39 

Collaborative Research 39 

Laboratory of Physiology 40 

Cancer Physiology Section 40 

Energy Metabolism Section 41 

Physical Biology Section 42 

Radiation Biology Section 43 

Office of the Chief 45 

Dermatology Branch 46 

Mycosis Fungoides Lymphoma 46 

Epidermal Growth and Reactivity 47 

Melanogenesis 47 

Endocrinology Branch 47 

Immunology Branch 48 

Metabolism Branch 49 

Amino Acid Transport 49 

Calcium Metabolism 49 

Porphyrin Metabolism 50 

Nucleic Acid 51 

Metabolism of Plasma Proteins 51 

Erythropoiesis 53 

Bilirubin Metabolism 54 

Surgery Branch 55 

Chemotherapy 63 

Acute Leukemia Service 64 

Solid Tumor Service 64 

"Life Island" 65 

Radiation Branch 65 

Clinical Branch, Baltimore Public Health Service Hospital 65 

Cell Separation 66 

Conclusion 66 

Laboratory of Chemical Pharmacology 66 

Etiology 69 

Biology Branch — 70 

Chemistry Branch 73 

Viral Oncology 75 

Viral Biology Branch 77 

Electron Microscopy Section 77 

Microbiology Section 77 

Virus Studies Section 78 

Virus Leukemia Section 78 

Viral Leukemia and Lymphoma 78 

Introduction 78 

Research and Activities in the Branch 79 

Special Virus-Leukemia Program 82 

NATIONAL HEART INSTITUTE 83 

Cardiology Branch 83 



CONTENTS IX 

Page 

Mechanics and Energetics of Miocardial Contraction: Isolated 

Heart Muscle 83 

Application of myocardial mechanics to the Intact Heart 86 

Contractile Properties of the Failing heart 89 

Isolated Heart Muscle and Subcellular Fractions 89 

Intact Dog Heart 90 

Clinical Studies 92 iX* 

On the Mechanism of Action of Digitalis 95 

On Circulatory Control of the Autonomic Nervous System 96 

Improvement of Cardiac Diagnostic Methods 98 

Clinical Studies 99 u^" 

Idiopathic Hypertrophic Subaortic Stenosis 99 

Other Clinical Studies 101 

Section of Clinical Biophysics 103 

Vascular Mechanics 103 

Myocardial Mechanics 106 

Lung Mechanics 106 

Surgery Branch 108 

Clinic of Surgery 108 

Laboratory of Clinical Biochemistry 112 

Amine Biogenesis ad Metabolism 112 

Collagen and Hydroxyproline 113 

Actinomycin Biosynthesis 114 

Cobamide Dependent Methionine Synthesis 114 

Formation of Formyl-Methionine sRNA 114 

Aromatic Hydroxylation 114 

Serine Metabolism 114 

Studies on Amino Acid Transport 115 

Peptides and Peptide Linkages 115 

Brain Nucleic Acid 115 

Bacterial Phenylalanine Hydroxylase and Its Cofactor 115 

Nitrogen Containing Lipids 115 

Enzyme Mechanisms 115 

Experimental Therapeutics Branch 116 

Biochemistry and Pharmacology of Aromatic Amines 116 

Studies of Selected Proteins 118 

Miscellaneous 120 

Laboratory of Technical Development 120 

Fluorescence Methods 120 

Ultramicro Methods 122 

Automation of Bacteria Counts and Antibiotic Sensitivity Tests 123 

Artificial Organs 123 

Fast Reaction Methods 124 

Chromatographic and Ultrasonic Methods 124 

Miscellaneous 125 

Laboratory of Cardiovascular Physiology 125 

Studies on Myocardial Mechanics 125 

Myocardial Oxygen Consumption 127 

Influence of Drugs and Electrolytes on the Heart 127 



CONTENTS 

Page 

Electrical Stimulation of the Heart 128 

Water and Salt Regulation 128 

Peripheral Circulation 128 

Kallikrein-Kininogen-kinin System 129 

Laboratory of Kidney and Electrolyte Metabolism 129 

Micropuncture Studies in the Kidney 129 

Isolated and Separated Tubules in Rabbits in Vitro 131 

Red Cell Studies 132 

Studies in Toad Bladder 133 

Chemical Characterization and Physiologic Significance of a 

Cardioglobulin System Present in Mammalian Plasma 134 

Renin-Angiotensin-Aldosterone System and Other Humoral 

Factors 134 

Laboratory of Biochemistry 135 

Sections on Enzymes 135 

Regulation of Divergent Biosynthetic Pathways of 

Metabolism 135 

Cumulative Feed-Back Inhibition of Glutamine Synthetase 135 

Uptake of Amino Acids by Isolated Cytoplasmic Membrane 

Preparations 138 

Metabolism of Amino Acids 139 

Reductive Deamination 139 

Lysine Fermentation 139 

One Carbon Metabolism 140 

Methane Fermentation 140 

Synthesis of Acetate from C0 2 141 

Cystathionine Biosynthesis and Trans-sulfuration 141 

Mechanism of Action of Vitamin Bi 2 Coenzymes 142 

Ethanolamine Diaminase 142 

Metabolism of Heterocyclic Compounds 143 

Hydroxylation of Nicotinic Acid 143 

Reduction of 6-Hydroxynicotinic Acid to 6-oxo-l,4,5,6- 

tetrahydronicotinic acid 143 

Chemical Synthesis of 6-Hydroxynicotinic Acid 143 

Section on Cellular Physiology 144 

Muscle Proteins: Myosin 144 

Extraction of Actinomyosin from Rabbit Muscle 144 

Radiation Damage in Proteins 145 

Protein Biosynthesis 145 

Nonphosphorylated Intermediates of Energy Transfer and 

Protein Biosynthesis 145 

Biochemistry and Cytology of Cell Transport 145 

Section on Comparative Biochemistry 146 

ACP Involvement in Lipid Metabolism 146 

Fatty Acid Biosynthesis 146 

Complex Lipid Biosynthesis 147 

Sterol Biosynthesis 147 

Mammalian ACP 147 

Structure of E. coli ACP 148 



CONTENTS XI 

Page 

Concentrations of ACP and CoA in E. coli 148 

ACP Hydrolase 149 

Clinical Endocrinology Branch 149 

Steroidogenesis by the Adrenal Cortex 149 

Calcium and Phosphorus Metabolism 150 

Renal Physiology 150 

Neuroendocrine Relationships 153 

Laboratory of Metabolism 153 

Section on Metabolism 154 

Mobilization and Utilization of Free Fatty Acids 154 

Metabolism of Adipose Tissue Studies in Vitro 154 

Utilization of FFA in Vivo and in Vitro 154 

FFA Metabolism in Cell Suspension of Ehrlich Ascites 

Tumor 154 

Studies on the Consequences of Rapid FFA Mobilization 155 

Factors Controlling Plasma Lipoprotein Concentration 155 

Metabolic Studies in Refsum's Syndrome, a New Lipid 

Storage Disease 156 

Clinical Studies 156 

Animal Studies 157 

Lymphatic Absorption of Lipids 158 

Section on Molecular Diseases 159 

Hyperlipoproteinemia 159 

Structure and Function of Lipoproteins 160 

Tissue Lipidoses 160 

Protein Structure and Function 160 

Section on Chemistry 161 

Laboratory of Biochemical Genetics 162 

Nucleotide Sequences of RNA Codons 162 

Mechanism of Codon Recognition 163 

Characteristics of Synonym RNA Codons 163 

Codon Recognition on 30 S Ribosomes 163 

Attachment of mRNA to Ribosomes 163 

Template Activity of Modified RNA Codons 163 

Universality of the Code 164 

Regulatory Mechanisms Dependent on Viral Infection 164 

Laboratory of Chemical Pharmacology 165 

Adrenergic Neurochemical Transducer 165 

Application of Steady State Kinetics 165 

Turnover Rates and Times of Catecholamines and 5HT 165 

Results of Studies of Turnover Rates 166 

Kinetics of Storage 166 

Recapture Mechanism 166 

Mechanism of NE Release by Nerve Stimulation 167 

Electrolyte Requirements for NE Storage and Uptake 167 

Kinetics of Reserpine-Induced Release of Biogenic Amines 168 

Relationship of Pharmacologic Effects of Reserpine to 

Effects on 5HT Storage 169 

False Adrenergic Transmitters 169 



XU CONTENTS 

Page 
Relationship of Pharmacologic Effects of False 

Transmitters to Rate of NE Efflux 169 

Effects of Desmethylimipramine (DMI) on Adrenergic 

Neurons 169 

The Serotonergic Transducer 170 

Role of Serotonin (5HT) 170 

Sympathetic Target Sites 170 

Adipose Tissue Transducer System 170 

Interaction of Sympathetic and Hormonal Systems 171 

Adrenergic Blocking Agents 171 

Electrolyte Requirements 171 

Studies on Chemical-Induced Shock 172 

The Nonmast Cell Histamine Transducer 172 

Selective Labeling of Nonmast Cell Histamine 172 

Distribution and Fate of Injected HVHistamine 173 

Release of HVBistamine 173 

Synthesis of Histamine 173 

Factors that Affect Drug Action 174 

Passage of Substances Across Membranes 174 

Enzymatic Mechanism of Membrane Transport 174 

Enzymatic Mechanisms of Drug Metabolism 175 

Mechanism of Teratogenesis of Thalidomide 175 

Pharmacogenetics . 176 

Clinical Studies with Desmethylimipramine 176 

Development of New Methods of Analysis 177 

NATIONAL INSTITUTE OF ALLERGY AND 

INFECTIOUS DISEASES 179 

Introduction 179 

Laboratory of Clinical Investigations 180 

Transmission Dynamics in Respiratory Infections 180 

Antibody in Nasal Secretions and Serum 181 

Chilling and Respiratory Infection 181 

Adenovirus Soluble Antigens 181 

Influenza 181 

Mechanisms of Fever and Host Responses 181 

Systemic Fungus Infection 182 

Leprosy 182 

Laboratory of Infectious Diseases 182 

Adenoviruses as Oncogenic Agents 182 

Tumor and T Antigens as Possible Determinants of 

Adenovirus Oncogenesis 182 

Adenovirus-SV40 Hybrids 183 

Adenovirus-Associated Viruses (AAV's) 183 

Leukemia 183 

Vaccine Against Mycoplasma Pneumoniae 183 

Mycoplasma Epidemiology 183 

Mycoplasma — Fundamental Studies 183 

Host Resistance to Respiratory Disease 184 

Studies in Oceania 184 



CONTENTS Xiii 

Page 

Rubella Virus 184 

Bacterial Metabolism and Physiology 185 

Histoplasmosis 185 

Laboratory of Biology of Viruses 185 

Viral Structure 186 

Cell Response to Viral Infection 186 

Viral Oncogenesis 186 

Genetic Relatedness 186 

Laboratory of Tropical Virology 186 

Middle America Research Unit (MARU) 187 

Hemorrhagic Fever 187 

Arbovirus Studies 187 

Laboratory of Germfree Animal Research 187 

Immunoglobulins in Germfree Mice 188 

Genetic Factors May Regulate Immune Tissue Destruction 188 

Clostridium Perfringens Lethal to Germfree Guinea Pigs 188 

Severity of Amoebic Infections Influenced by Associated 

Bacteria 188 

Low Serum Lysozyme Levels in Germfree and Ex-Germfree 

Rats 189 

Immunoglobulins Quantitated in Human Malaria 189 

Laboratory of Parasitic Diseases 189 

Schistosomiasis 189 

Amebiasis and Trichomoniasis 190 

Glucose and Glycerol Utilization in Parasites 191 

Laboratory of Parasite Chemotherapy 191 

Malaria — Human 191 

Malaria — Simian 192 

Immunological Studies 193 

Laboratory of Immunology 193 

Autoimmunity 193 

Transplantation Immunology 194 

Hypersensitivity 194 

Mitogens 194 

Rocky Mountain Laboratory 195 

Selected Zoonoses of Regional Importance 195 

Rickettsial Diseases 195 

Transmission of Disease Agents by Vectors 196 

Encephalitides and Tick-Borne Diseases 197 

Psittacosis — Lymphogranuloma-Trachoma (PLT) 197 

Chronic Progressive Viral Disease 198 

Allergy and Immunology 198 

Immunoprophylaxis Against Tuberculosis 199 

Bordetella Pertussis Antigens 200 

Endotoxins 200 

Microbial Proteins and Nucleic Acids 200 

Biology of Microbial Agents in Arthropods 201 

Laboratory of Bacterial Diseases 201 

Mycoplasma 201 

Brucellosis 202 



XIV CONTENTS 

Page 
NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC 

DISEASES 203 

Introduction 203 

Laboratory of Nutrition and Endocrinology 204 

Studies in Folic Acid 204 

Large-Scale Processing of Biological Materials 205 

Studies in Experimental Nutrition 205 

Study of Protein Hormones 205 

Diabetes and Fat Metabolism 205 

Action on Isolated Fat Cells 205 

Fat Soluble Vitamins and Related Substances 206 

Reversible Chemical Modification of Cytochrome 206 

Purine-Pyrimidine Balance and Hepatic Fat Metabolism 206 

Laboratory of Biochemistry and Metabolism 206 

Polysaccharide Metabolism 206 

Bacterial Systems 206 

Animal Systems 207 

Glycoprotein Studies 207 

Antibodies 207 

Liver, Oviduct and Mastocytoma Glucoproteins 208 

Ribonucleic Acid 208 

Studies on RNase II 208 

Studies on RNase I 208 

Thionucleotide Studies 209 

Biochemical Studies of Lysogeny 209 

Enzymes Near the Bacterial Surface 209 

Surface Enzymes; Removal by Osmotic Shock 209 

Enzymatic Utilization of Model Compounds in Bacterial 

Systems 210 

Phenotypic Expression of Genetic Information 210 

Galactose in E. coli 210 

Hormone-induced Differentiation, Mouse Gland 211 

Reduction of Protein Disulfide Bonds 211 

Laboratory of Chemistry 212 

Section on Biochemical Mechanisms 212 

Electrolytic Cleavage of Tyrosyl-Peptide Bonds 212 

Synthetic Models for Hydrolytic Enzymes 212 

Oxidation Mechanisms in Metabolic Processes 212 

Section on Carbohydrates 212 

Studies on Presumed Ester Linkage in Glycoproteins 212 

iV,N-Diacylhexosamines and Conformation of Amino Sugars 213 

Studies Synthesis of Polyvinyl Glycosides 213 

Higher-Carbon Sugars 213 

Thio Sugars 214 

Amino Sugars 214 

Section on Medicinal Chemistry 214 

Evaluation of Analgesics 214 

In Vitro Screening for Anti-Inflammatory Properties 214 

Optical Resolutions of Benzomorphans 214 



CONTENTS XV 

Page 

Abnormal Stevens Rearrangement 215 

9-Acetoxy and -hydroxy-benzomorphans 215 

New Aromatization for Z>eta-tetralones 215 

Schiff-Bases and Oxazolidines 215 

Potential Aldolase Inhibitors 215 

Immunologic Studies in Clinical Hematology 215 

Dihydrocodeinone Isomers 215 

Polyethylenimine Studies 216 

Synthesis of 4-p-methoxy-l-vinyl-2-pyrrolidone 216 

Napth- and Benz-3,4-dihydro-oxazines 216 

Section on Microanalytical Services and Instrumentation 216 

Section on Steroids 216 

Study of Alkaloids from Solanum congestiflorum 216 

Synthesis and Reactions of Heteroisteroids 216 

Mass Spectrometry of Steroidal Glycosides 217 

Sterols from Parasites 217 

Photoreduction of fieta-Estradiol 217 

Chemistry of Iresine Celosia 217 

Sterol Biosynthesis in Plants 217 

Metabolism of C 14 -labeled Steroids 217 

Adrenal Metabolism of the Gerbil 217 

Biosynthesis and Metabolism of Biogenic Amines 218 

Section on Metabolites 218 

Batrachotoxin, Strongest Venom Known 218 

Pharmacology and Toxicology of Batrachotoxin 218 

Venoms of Other Amphibians _, 218 

Synthesis and Biosynthesis of Dehydrobufotenine 218 

Reactivity toward N-Bromosuccinimide of Tryptophan 219 

Novel Photocyclization of Tryptophan Derivatives ^ iy 

Photoreductions of Tryptophan " 

Novel Photocyclization in Tyrosine and Tyramine 9 

Photoreduction of Histidine 219 

Binding Characteristics of Polyuridylic Acid 219 

Template Activity of Uridyllic Acid 220 

Mechanism of Two-Step Reduction of Thymidine 220 

Stereoisomerism in Photoreduction of Thymidine 220 

Stereochemistry of 3-Methylproline 220 

Proton Magnetic Resonance Spectra of 3,4-Dehydroproline 

Derivatives 220 

Proline Analogs, Effects upon Biosynthesis of Actinomycins 221 

Analogs and Homologs of Proline and Hydroxyproline 221 

Synthesis of Erythro-gamma-Hydroxy-L-\ysme 221 

Synthesis and Metabolism of 6-Hydroxycatecholamines 221 

Chemorelease of Norepinephrine from Mouse Hearts 222 

Drugs Affecting the Nervous Systems 222 

Depletion of Norepinephrine- 3 H from Heart by Alpha- 

Methyl-m-Tyrosine 222 

Preparation of Gramicidin A, B, and C 222 

Release of Kinin Activity from Human Kininogens 222 



XVI CONTENTS 

Page 

Selective Cleavage of Cysteine Peptide Bonds 223 

Cleavage of Cysteine Peptide Bonds in Insulin 223 

Antibiotic Nisin 223 

Active Carboxy Group of Pepsin 223 

Improvement in Methodology of Amino Acid Analysis 224 

Laboratory of Experimental Pathology 224 

Introduction 224 

Pathology of Rheumatic Diseases 225 

Animal Joint Lubrication 225 

Cytogenetics 226 

Experiments on Reutilization of Chromosomal DNA 226 

Meiotic Chromosome Studies in Human Male 226 

Characterization of Enzymes in Histochemical Systems 226 

Histochemical Localization of ATPase Activity 226 

Studies in Oxidative Phosphorylation 227 

Mechanism of Protection by 5-HT in Tryptophan Deficiency 227 

Fluorescein Studies 227 

Localization of Antigens in Tissues 227 

Localization of Prolactin in Rat Pituitary 228 

Localization of Prolactin in Fundulus heteroclitus, L. 228 

Studies on Nucleic Acids and Protein Synthesis 228 

Studies on Soluble Ribonucleic Acid 229 

Electron Microscope Studies 229 

Amyloidosis 229 

Fine Structure of Rabbit Marrow Cells 229 

Myeloid Elements 229 

Studies of Human Lymphocytes 229 

Fine Structural Characterization of Neuromelanin 230 

Neuromelanin 230 

Experimental Porphyria 230 

Cytochemistry of Bacterial Phosphatases 230 

Temperature Effect on Lipids of Leishmania 230 

Mouse Thyroid Gland Cytology 231 

Thyroid Parafollicular cells 231 

Mucosaccharide Histochemistry 231 

Microspectrophotometric Studies of Basic Protein 232 

Iron Staining of Acid Mucosubstances 232 

Studies on Endocarditis • 232 

Studies on Dimethyl Sulfoxide 232 

Studies on High Altitude Hypoxia 233 

Studies on Hamycin 233 

Cycasin Research 233 

Studies in Germfree Rats 233 

Mutagenic Effect 234 

Transplacental Induction of Tumors 234 

Prevention of Acute Toxicity of Cycasin 234 

Transplantation of Cycasin Induced Neoplasms 234 

Laboratory of Chemical Biology 234 

Structure-Function Relationships in Proteins 234 



CONTENTS XVU 

Page 
Structure, Function, and Synthesis of a Nuclease from 

Staphlococcus aureus 236 

Mechanism of Control of Biosynthesis of Histidine in 

Salmonella 237 

Biochemical Studies Preliminary to Investigation of Genetic 

Defects in Higher Organisms, including Man 238 

Laboratory of Biochemical Pharmacology 238 

Studies with EDTA-treated E. coli 238 

Inhibitory Effect on Actinomycin 238 

Surface Changes in EDTA-treated E. coli 239 

Bacteriophage Studies 239 

EDTA-treated E. coli 239 

Lysogenic Induction 239 

Ribosome Structure and Function 239 

Amine Studies 239 

Polysaccharides and Glycoproteins 240 

Control of Enzyme Levels in Mammalian Tissues 240 

Burn-Shock Studies 240 

Burns 240 

Tourniquet Trauma 240 

Sulfur-containing Compounds 240 

Leprosy Studies 240 

Laboratory of Physical Biology 241 

Molecular Structure 241 

Surface Phenomena 242 

Physiology and Biochemistry 242 

Excitation and Coupling 242 

Biological Interactions 243 

Biological Ultrastructure 243 

Cellular Biology 244 

Laboratory of Biophysical Chemistry 244 

Laboratory of Molecular Biology 246 

Structure and Function of Glutamate Dehydrogenase 246 

Enzyme Induction in Bacteria 246 

Cysteine Biosynthesis 246 

Enzyme Induction by Steroid Hormones 246 

Active Transport of Amino Acids in Salmonella typhimurium 247 

Amino Acid Incorporation into Protein In Vitro 247 

Investigations of Nucleic Acids and Related Substances 247 

X-ray Diffraction studies on Proteins 247 

A Search for DNA Recombination In Vitro 248 

Structure of Polyadenylic Acid at pH 7 248 

Optical Properties of Nucleic Acids 248 

Interaction of DNA with Polycations 248 

Internal Proteins of Bacteriophage T2 248 

Glucosylation and Kinetics of Endonuclease Degradation of DNA 249 

Viscosity Studies of DNA of Bacteriophage Mutants 249 

Chemistry and Genetics of Hemoglobin and Other Proteins 249 

Biochemical Control Mechanisms in Histidine Biosyntheses 250 



XV111 CONTENTS 

Page 

Mechanism of Lambda Bacteriophage Induction 250 

Mathematical Research Branch 251 

Clinical Investigations 253 

Arthritis and Rheumatism Branch 254 

Studies of Natural History of Disease 254 

Lymphocyte Function in Sjogren's Syndrome 254 

Immunological Reactivity in the Elderly 255 

Variations in Rheumatoid Factor Titer 255 

Clinical and Metabolic Evaluation of Cystinosis 255 

Therapeutic Studies 255 

Prophylactic Synovectomy in Rheumatoid Arthritis 255 

Melphalan in Systemic Amyloidosis 255 

Lymph Drainage in Systemic Lupus Erythematosus 255 

Mechanisms of Disease 255 

Purine Biosynthetic Pathways 255 

Induced Renal Retention of Uric Acid 255 

Role of Infection in Rheumatic Disease 256 

Experimental Amyloidosis 256 

Ribosomes in Hormonal Control of Protein Synthesis 256 

Mechanisms of Action of Adrenocorticotrophic Hormone 

Studies on Immunoglobulins 256 

Antibody Synthesis in the Spleen ^6 

Macroglobulin Subchains 2 ^6 

Macroglobulin Catabolism 256 

Studies on Canine Immunoglobulins 257 

Miscellaneous 257 

Carbon-Fluorine Bonds in Biology 257 

Metabolic Diseases Branch 257 

Research on Human Physiology and Nutrition 257 

Total Energy Balance 257 

Effects of Heat Stress on Potassium Balance 257 

Mechanisms of Water and Electrolyte Secretion by Human 

Sweat Gland 257 

Calcium Metabolism 257 

Bone Cell Metabolism 258 

Chemistry and Physiology of Parathyroid Hormone 258 

Action of Thyrocalcitonin 258 

Studies of the Small Intestine 258 

Whipple's Intestinal Lipodystrophy 258 

Other Diseases 259 

Intermediary Metabolism of the Intestinal Mucosa 259 

Ultrastructure of the Human Intestinal Mucosa 259 

Inborn Errors of Metabolism 260 

Homocystinuria 260 

Cystathioninuria 260 

A New Disorder 260 

Intermediary Metabolism 260 

Clinical Endocrinology Branch 261 

Thyroid Biochemistry 261 



CONTENTS XIX 

Page 

Iodide Transport 261 

Iodination Reactions and Thyroxine Synthesis 261 

Iodoproteins 262 

Protein Synthesis in the Thyroid Gland 262 

Proteolytic Enzymes in the Thyroid Gland 262 

Measurement of Iodocompounds in Biological Fluids 262 

Pituitary Hormones 263 

Growth Hormone 263 

Gonadotropins 263 

Thyrotropin 263 

Vasopressin 263 

Insulin, Glucagon and Carbohydrate Metabolism 263 

Regulation of Protein Metabolism 263 

Regulation of hepatic carbohydrate metabolism 264 

Glucose Transport 264 

Galactose Metabolism and Galactosemia 264 

Amino Acid Transport 264 

Protein Structure 265 

Pediatric Metabolism Branch 265 

Immunological and Biochemical Investigations 265 

Inborn Error of Mucopolysaccharide Metabolism in Cystic 

Fibrosis 265 

Serologic Reactions in Patient with CF 266 

Pseudomonas aeruginosa and its Slime in Relation to 

Pathogenesis of Cystic Fibrosis 266 

Structure-Function Studies with Electron Microscope 267 

Effect of Aldosterone on Sweat Gland and Renal Function 

in Normal Subjects, Patients with CF, and Carriers 267 

Normal adults and children 267 

Patients with Cystic Fibrosis 267 

Other Investigations in Cystic Fibrosis 268 

Pregnancy in CF 268 

New Syndromes of Pancreatic Deficiency 268 

Albumin Turnover Studies in CF 268 

Growth, Development, and sexual maturation in Patients 

with Cystic Fibrosis 269 

Clinical Hematology Branch 269 

Immunology of Blood Cell Deficiencies 269 

Idiopathic Thrombocytopenic Purpura 269 

Reticuloendothelial Blockade in Sequestration of 

Immunologically Altered Cells 269 

Etiology of Transfusion Anuria 269 

Post-transfusion Purpura 270 

Isoimmune Neonatal Purpura 270 

Transplantation Immunity 270 

Histocompatibility in Human Homograft pairs 271 

Immunologic Therapy of Malignancies 271 

Immunological Considerations Attending Platelet 

Transfusions 272 



XX CONTENTS 

Page 

Blood Coagulation and Diseases of Hemorrhage and Thrombosis 272 

Spleen Controlling Level of Anti-hemophilic Factor 272 

Evaluation, new Plasma Fraction in treatment of Classical 

Hemophilia 273 

Basis for Thrombocytopenia in Malaria 273 

Diseases of Megakaryocyte dysfunction 274 

Platelet Physiology 274 

Unusual Hemorrhagic Disorders 274 

NATIONAL INSTITUTE OF CHILD HEALTH AND 

HUMAN DEVELOPMENT 275 

Introduction 275 

Reproduction Program 275 

Growth and Development Program 276 

Aging Program 276 

Mental Retardation Program 277 

Intramural Programs 277 

Thyroid-stimulating Hormone (TSH) 278 

Luteinizing Hormone (LH) 278 

Melanocyte-stimulating Hormone (MSH) 278 

Growth Hormone 279 

Laboratory of Biology - 279 

Growth and Development Program 280 

Aging Program 281 

Summary 281 

Research at Bethesda 284 

Gerontology Branch 284 

Aging in the Human 285 

Human Performance Section 285 

Section on Experimental Psychology 285 

Metabolism Section 286 

Nutritional Biochemistry Section 287 

Pulmonary Physiology Section 287 

Renal Section 288 

Endocrinology Section 289 

Metabolism Section 290 

Biology of Aging 290 

Section on Animal Behavior 290 

Cardiovascular Section 291 

Section on Nutritional Biochemistry 291 

Section on Molecular Biology 292 

Section on Comparative Biochemistry 293 

Section on Cellular and Comparative Physiology 294 

Biophysics Section 296 

Mental Retardation Program 297 

Direct Research 297 

Laboratory of Biomedical Sciences 298 

Biochemical Investigations 298 

Cytogenetic Investigations 298 

Neurophysiology Investigation 298 



CONTENTS XXI 

Page 

Children's Diagnostic and Study Unit 299 

NATIONAL INSTITUTE OF DENTAL RESEARCH 301 

Introduction 301 

Dental Caries 302 

Periodontal Disease 306 

Growth and Development 308 

Collagen, Elastin, and Calcification 311 

Cell Biology and Enzyme Chemistry 312 

Virology, Immunology, and Microbial Physiology 314 

Oral Soft Tissue Lesions, Clinical Diagnosis, Treatment 317 

Laboratory of Microbiology 319 

Immunology 319 

Dental Caries 321 

Virology 322 

Microbial Physiology 323 

Systematic Microbiology 324 

Laboratory of Histology and Pathology 324 

Microstructural Characteristics of Enamel 324 

Crystallographic Studies of Synthetic and Biological Phosphates 325 

Histochemical and Chemical Studies of Connective Tissues 325 

Experimental Pathology 326 

Bacterial and Macromolecular Structure 326 

Laboratory of Biochemistry , 326 

Enzyme Chemistry 326 

Enzyme Structure and Mechanism of Action 327 

Methyl Groups of sRNA 327 

Phosphate and Dental Caries 327 

Prenatal and Fetal Development Factors Influencing Oral 

Disease 328 

Protein Chemistry 328 

Fluoride Metabolism 329 

Salivary Biochemistry 329 

Epidemiology and Biometry Branch 329 

Nutrition Surveys 330 

Dental Caries 330 

Periodontal Diseases 331 

Occlusal Anomalies 331 

Collateral Projects 331 

Clinical Studies 331 

Dental Services Branch 333 

Work with the National Cancer Institute 333 

Work with the National Institute of Arthritis and Metabolic 

Diseases 334 

Work with the National Heart Institute 334 

Oral Medicine and Surgery Branch 334 

Studies of the Human Dental Pulp 334 

Anesthesia Studies 335 

Dental Caries 336 

Studies of Soft Tissue Lesions 336 



XXii CONTENTS 

Page 

Clinical Periodontal Studies 337 

Animal Periodontal Studies 337 

Oral Pathology Investigations 338 

Oral and Pharyngeal Development Section 339 

Basic Anatomical Studies 339 

Basics Physiological Studies 339 

Human Genetics Branch 340 

Congenital Malformations 340 

Genetic Factors in Dental Caries 341 

Biochemical Genetic Defects 341 

Effects of Radiation Exposure . 341 

Biochemistry of Normal Variation and Cellular Biology 341 

Cellular Biology and Cytogenetics Section 342 

Future Studies 342 

NATIONAL INSTITUTE OF MENTAL HEALTH 343 

Introduction 343 

Clinical Investigations 344 

Addiction Research Center 346 

General 346 

Administrative 347 

New Analgesics and Pathological Processes 348 

Acute and Chronic Intoxication with Drugs 349 

Marihuana Group 349 

Clinical Studies of Intoxication 349 

Biochemistry of Addiction 350 

Neurophysiology and Neuropharmacology of Chronic 

Intoxication 350 

Effect of Chloropromazine on Barbiturate Withdrawal 350 

Barbiturate Withdrawal in rats 350 

Free Choice Between Tap Water and 5% Alcohol in rats 351 

Sodium Barbital on Electroconvulsive Threshold Elevation 351 

Psychological Studies on Addiction 351 

Mode of Action of Central Nervous System Depressants 352 

Conditioning Factors in Opiate Addiction and Habituation 352 

Experimental Studies with Human Subjects and Studies on 

Learning 353 

Social Science Section 354 

Biochemical Pharmacology 355 

Laboratory of Socio-Environmental Studies 355 

Studies of Social Context of Personality Development 355 

Social Class, Occupation, Parental Values 355 

Values and Behavior 356 

Maternal Care and Child Behavior in Japan 356 

Children's Orientation to Health and Illness 357 

Experimental Modification of Children's Behavior 357 

Review of Research on Social Development 357 

Population Genetics 357 

Social Psychological Correlates of Occupation 358 

Studies of Mental Disorder 358 



CONTENTS XX111 

Page 

Quantitative Studies of Psychology and Schizophrenia 358 

Social Epidemiology of Schizophrenia 358 

Studies of Methodology of the Social Sciences 359 

Assessment of Methods of Developmental Psychology 359 

Methodological Principles of Survey Analysis 360 

Laboratory of Neurophysiology 361 

Laboratory of Psychology 363 

Section on Early Learning and Development 363 

Section on Personality 363 

Office of the Chief 363 

Section on Perception 363 

Section on Higher Thought Processes 363 

Section on Neuropsychology 363 

Development of Behavioral Function 364 

Neonatal Learning 364 

Social Behavior 364 

Dr. Gewirtz' program, Research on Impact of Environment 365 

Intellectual Development 367 

Analysis of Developed Functions 367 

Communication 369 

Thought Process 369 

Variables Affecting Biological Systems 370 

Biological Substrate of Behavioral Functions 370 

Cortical Mechanisms in Sensation and Perception 371 

Vision 371 

Audition 371 

Somesthesis 371 

Visuomotor Coordination 372 

Cortical Mechanisms in Problem Solving 372 

Cortical-subcortical Relations and Regulation of Behavior 372 

Destroyers of Behavioral Function 373 

Crowding 373 

Schizophrenia 374 

Enhancers of Behavioral Function 378 

Theoretical, Methodological and Technical Advances 379 

Administrative Aspects of Laboratory 380 

Child Research Branch 382 

Clinical Investigations 382 

Introduction 382 

Patterns of Middle Class Early Marriage 384 

Early Parent-Infant Patterns 385 

Studies of Individual Differences in Infants 385 

Toward a Theory of Marital Experience 386 

New Developments 386 

Adult Psychiatry Branch 387 

Family Studies 387 

Intrafamilial Relationships and Transactions 387 

Study of Psychotherapy, Especially Family Therapy 389 

Cross-Cultural Family Studies 390 



XXiv CONTENTS 

Page 

Family Development 390 

Experimental Ego Psychology 390 

Clinical and Conceptual Studies of Schizophrenia 390 

Personality Development 391 

Twin and Sibling Studies 392 

Psychosomatic Studies 394 

Psychophysiology of Sleep 398 

Laboratory of General and Comparative Biochemistry 400 

Section on Proteins 400 

Section on Cellular Regulatory Mechanisms 401 

Section on Alkaloid Biosynthesis 402 

Section on Technical Development 402 

Projects Completed 403 

Projects in Process 403 

Projects Abandoned 404 

Section on Technical Development Projects 404 

Projects Initiated within the Section 404 

LINC Programs in Current Active Use 404 

LINC Programs in Preparation 404 

Laboratory of Neurochemistry 404 

Laboratory of Clinical Science 406 

Section on Cerebral Metabolism 408 

Section on Pharmacology 409 

Section on Medicine 411 

Section on Biochemistry 412 

Section on Physiology 414 

Pyramidal Tract Activity and Voluntary Movement 414 

Brain Mechanisms in Eye Movement Information 415 

Significance for Mental Health Research 415 

Section on Psychiatry 416 

Unit on Psychosomatics 418 

Nervous and Circulatory Systems 418 

Diet and Catecholamine Excretion 418 

General Health of Families 419 

Observations in Psychiatric Illness 419 

Clinical Neuropharmacology Research Center 419 

Laboratory of Neurobiology 422 

NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES 

AND BLINDNESS 425 

Introduction 425 

Ophthalmology 425 

Medical Neurology 426 

Surgical Neurology 426 

Medical Neurology Branch 426 

Clinical Investigation Program 426 

Myopathies 427 

Episodic Weakness 427 

Myasthenia Gravis 427 

Amyotrophic Lateral Sclerosis 428 



CONTENTS XXV 

Page 

Methodology 428 

Neuroradiology Section 428 

Radiographic Diagnosis 428 

Radiation Dosimetry 429 

Isotopic Diagnosis 429 

Neuropharmacology Section 429 

Branch of Surgical Neurology 430 

Developmental Defects 430 

Epilepsy 431 

Involuntary Movements 432 

Brain Tumor 433 

Cerebral Edema 433 

Cerebral Trauma 434 

Language and Memory 435 

Effects of Low Temperatures 435 

Neurosurgical Monitoring 436 

Microbial Analysis of Neurosurgical Environment 436 

Summary 437 

Branch of Ophthalmology 437 

Laboratory Investigations 438 

Retina 438 

Cornea and Vitreous 439 

Choroid 440 

Lens 440 

Intraocular Pressure 440 

Clinical Investigations 441 

Retina 441 

Uvea 441 

Glaucoma 441 

Ocular Changes in Systemic Disorders 442 

Branch of Electroencephalography and Clinical Neurophysiology 442 

Clinical Diagnostic Service 442 

Research Activity 443 

Other Activities and Organizational Aspects 444 

Program Considered for Near Future 445 

Laboratory of Neuroanatomical Sciences 445 

Introduction 445 

Analyses of Structure 446 

Cytology 446 

Auditory and Vestibular Systems 446 

Olfactory System 446 

Automatic Nerve Endings 446 

Pathways of Transport in Brain 447 

Cytoarchitecture 447 

Auditory System 447 

Muscle Spindle 448 

Interactions in and among Sensory, Central, and Motor 

Systems 448 

Trophic Interactions 448 



XXVI CONTENTS 

Page 

Nerve-Muscle Relationships 448 

Visual System 449 

Morphogenesis 450 

Auditory System 450 

Regeneration 450 

Future of the Program 450 

Laboratory of Neuropathology 450 

Section on Experimental Neuropathology 450 

Perfection of Method of Preservation 450 

Identification of Artifactual Changes 451 

Pathologic Neuronal Manifestations 451 

Identification of Cell Types 451 

Laboratory of Neurophysiology 451 

Laboratory of Biophysics 452 

Laboratory of Neurochemistry 453 

Laboratory of Molecular Biology 455 

Structure and Alteration of Nucleic Acids 455 

Use of Transforming DNA 455 

Effect of Hydrogen Peroxide on DNA 456 

Effect of Combining Mutagenic and Inactivating DNA alter- 
ations 457 

Genetics Analysis of Virus Included Mutants of E. coli 457 

Studies on the Structure of Chromosomes 457 

Control Mechanisms and Differentiation 457 

Depression of Alanine Dehydrogenase in Bacillus subtilis by 

Internal Induction 457 

Lack of Sporulation in Cytochrome alpha-Deficient mutants 

of B. subtilis 458 

New Lysyl sRNA Produced during Sporulation 458 

Initiation of Germination 458 

Laboratory of Perinatal Physiology 458 

DIVISION OF BIOLOGICS STANDARDS 467 

Introduction 467 

Research and Development 468 

Section on Experimental Virology , 468 

Laboratory of Bacterial Products 469 

Cholera Vaccine and Related Studies 469 

Pertussis Vaccine 469 

Tetanus Toxoids 469 

Pleuropneumonia-like Organisms and Mycoplasma 470 

Mycoplasma orale 470 

Tuberculin 470 

Poison Ivy 470 

Laboratory of Biophysics and Biochemistry 470 

Microbial Biophysics 470 

Analytical Chemistry 471 

Virus Characterization 471 

Laboratory of Blood and Blood Products 471 

Laboratory of Pathology 472 



CONTENTS XXVH 

Page 

Neurovirulence Testing 473 

Oncogenesis and Pathogenesis of Infectious Agents 473 

Neuro- Anatomy 473 

Laboratory of Viral Immunology 473 

Rubella 473 

Viral Genetics 474 

Mumps 474 

Vaccinia 474 

Laboratory of Virology and Rickettsiology 474 

Arboviruses 475 

Avian Leukosis 475 

Adenoviruses 475 

Influenza 476 

Rickettsiae 476 

Tissue Culture 476 

Tissue Culture Section 476 

Paolins 476 

Hepatitus 477 

Contract Operations 477 



NATIONAL CANCER INSTITUTE 



GENERAL LABORATORIES AND CLINICS 

Introduction 

The reorganization of the National Cancer In- 
stitute during fiscal year 1966, dividing the 
Institute activities into 4 program areas, in- 
corporates within the General Laboratories 
and Clinics (GL&C) program most of the Insti- 
tute's intramural research, although the for- 
mer Laboratory of Viral Oncology is now 
within the Etiology program area, and the 
Radiation Branch and Medicine Branch are 
now within the Chemotherapy program area. 
Currently, the GL&C consists of 4 Laborator- 
ies (Biochemistry, Biology, Physiology, and 
Pathology), 6 Clinical Branches (Surgery, Me- 
tabolism, Immunology, Endocrinology, Derma- 
tology, and Pathologic Anatomy), and one 
section reporting directly to the Office of the 
Scientific Director (Macromolecular Biology 
Section), the latter having been within the 
Laboratry of Viral Oncology prior to reor- 
ganization. 

Although the major targeted research activi- 
ties in tumor virology, radiation biology, and 
chemotherapy are now pursued within the 
other program areas, there remains substan- 
tial representation of these activities within 
the GL&C, particularly tumor virology and 
radiation biology. There is comparatively lit- 
tle representation of chemotherapy per se. 
However, the clinical investigations of chemo- 
therapy are a direct operational activity of the 
Medicine Branch pursued in the Clinical Cen- 
ter, which geographically provides for easy 
collaborative and cooperative clinical investi- 
gations between the clinical branches of the 
GL&C and the Medicine Branch of the Chemo- 
therapy area. 

The organizational identity of the more tar- 
geted programs of the Institute in turn identi- 



fies the research program of the GL&C as now 
consisting of the conduct of less targeted basic 
research but oriented toward developing 
knowledge leading to the final understanding 
of the nature of cancer, its causes, its treat- 
ment and its prevention. Superimposed upon 
this are identifiable characteristics of the re- 
search program which have evolved over the 
years from direction exerted by the scientists 
within the GL&C and the particular program 
interests generated within the Institute. Some 
identifying characteristics include a high de- 
gree of emphasis and strength in biochemis- 
try, virology, carcinogenesis and immunology. 
Beyond these areas of emphasis there is, how- 
ever, a wide range of interests represented 
within the GL&C which includes significant 
research activity in electron micrographic 
anatomy, in vitro and in vivo tissue culture 
studies, endocrinology, mathematical biology, 
genetics, cellular kinetics and anticarcinogen- 
esis. It should be noted there has been little 
emphasis to date on developmental biology, 
membrane biology, mathematical and theoreti- 
cal biology, lipid chemistry and connective tis- 
sue chemistry. 

The staff of the GL&C now consists of ap- 
proximately 500 persons, of which approxi- 
mately 200 are professional level staff. 

The following is an abbreviated account of 
selective research activities within the constit- 
uent laboratories and branches of the GL&C 
during fiscal year 1966. More detailed descrip- 
tion can be found in the individual reports of 
each area. 

The Laboratories 

The MACROMOLECULAR BIOLOGY SECTION, 

within the Laboratory of Viral Oncology prior 
to the NCI reorganization during the past year, 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



is now a separate research unit not organiza- 
tionally attached to a laboratory or branch. 

Past and present research interests of the 
Section have involved the interaction of large 
molecules, including polysaccharides, proteins 
and nucleic acid polymers. The research course 
projected has some additional orientation to 
the process of malignant transformation of 
cells and tissue recognition phenomenon oc- 
curring during the process of differentiation, 
where the techniques and knowledge of the 
Section seem to be applicable. 

During the past year the active enzyme, B- 
galactosidase, of E. coli has been shown to be 
a tetramer, with a molecular weight of 540,000 
and a sedimentation coefficient of 16s. The in- 
active unit is found to be a dimer, molecular 
weight of 220,000 and sedimentation coemicient 
of 10s. Denaturation of the enzyme with 5-6 
M urea has yielded a monomer unit with a 
sedimentation coefficient of 2s. 

The base sequence of DNA of normal rat 
liver and of rat hepatoma has not been found 
to possess significant differences, on the basis 
of optical properties identified in "Melting" 
profiles. 

Intact RNA was isolated from the Rauscher 
mouse leukemia virus and has been character- 
ized as a single-stranded molecule, with a mole- 
cular weight of 13x10°. The isolation of the 
molecule should permit studies to determine 
whether the isolated RNA possesses complete 
genetic information to effect malignant trans- 
formation in the absence of the intact virus 
particle. 

The electron microscope facility has been 
reactivated, following several months idleness, 
and will permit certain membrane studies con- 
sidered to be important in understanding 
transformation, cell recognition during contact 
inhibition, morphogenesis, etc. 

In the laboratory OF biochemistry research 
is highly oriented toward identifying biochem- 
ical changes associated with the cancer process. 
Most of the work, however, is applicable to 
areas and problems other than cancer. 

Metabolism of tumors, particularly in re- 
gard to glycolytic characteristics, received fo- 
cused, attention by several investigators. Re- 
cent work utilizing tumors in in vivo isolation 



has shown that tumors under such conditions 
utilize oxygen and glucose in proportionate 
amounts, that such tumors do not selectively 
utilize glucose in the absence of oxygen. An 
additional observation made on this isolated 
tumor system is that tumors metablizing high 
levels of both oxygen and glucose do not grow 
faster than when lower levels are utilized, but 
indeed may grow less rapidly. The destructive 
effects of hyperthermia on tumors in the in- 
tact animal seem to be greater than on normal 
tissue; this disparity is due in part to impair- 
ment of the tumor blood flow which secondar- 
ily may account for decreased glucose and 
oyxgen supply. 

Production of transplantable heptomas, pro- 
viding research material for some two dozen 
collaborative laboratories in the United States 
and abroad, is now accomplished .through a 
contract with a commercial laboratory (Mel- 
par). Considerable study of these tumors, 
however, continues at NCI and NIH. It has 
been found that many tumors, similar to nor- 
mal liver, are slow growing, have fewer tissue- 
specific antigens, and have a reduced capacity 
to incorporate thymidine into DNA. Some of 
the free polyribosomes will bind to membranes 
from normal liver but polyribosomes, originally 
bound to membranes of normal rat liver, do not 
bind to membranes from hepatomas. 

The chemical carcinogens used to produce 
hepatomas have also been used to induce blad- 
der tumors. The induction of such tumors re- 
quires low dietary vitamin B6. Therefore, an 
"anti-carcinogenic" effect of vitamin B6 may 
be identified and in this respect is similar to 
the anti-carcinogenic effect of vitamin A iden- 
tified in studies performed in the Pathologic 
Anatomy Branch. 

Controls of enzyme functions have been 
studied in rat mammary tumors, normal liv- 
ers and hepatomas. Primary mammary tumors 
from lactating hosts have been found to have 
a negligible capacity to produce lactose al- 
though it is known that mammary tumors 
possess the anzymes involved in lactose syn- 
thesis. This may be an example of the loss of 
ability of neoplastic cells to respond to influ- 
ence controlling functions of normal cells. 



NATIONAL CANCER INSTITUTE 



3 



Clones of hepatoma cell lines, containing 
either high catalase activity or low catalase 
activity, have been isolated from liver metas- 
tases when mixtures of tumor cell lines have 
been injected into the spleens of animals. 

A positive feedback mechanism has been 
identified in normal rat liver which involves 
the enzyme phosphoryl choline-cytidyl trans- 
ferase, a step in the synthesis of lecithin. It 
has been found that the enzyme is activated by 
degraded phospholipid, and inhibited by a poly- 
nucleotide. 

Studies on the site of synthesis of protein 
within the cell, and the intracellular distribu- 
tion of protein after synthesis, have shown 
that approximately half of the protein in 
plasma cell tumors is synthesized by the mi- 
cromosome fraction and mitochondria, and 
another half synthesized by the free polyribo- 
somes. The protein synthesized by the micro- 
somal fraction and mitochondria tends to re- 
main within these structures, whereas the 
greater portion of the protein synthesized by 
the free polyribosomes is distributed as soluble 
protein within the cytoplasm. The structural 
ribosomal proteins of plasma cell tumors of 
BALB/c mice have been found to consist of 
approximately 35 discrete protein bands elec- 
trophoretically. These bands have been found 
to be identical for 9 different tumors, even 
though each tumor secretes a different mye- 
loma protein. The ribosomal protein pattern of 
the tumor is the same as that of the normal 
liver of these mice. However, the ribosomal pro- 
tein patterns of mouse liver, rat liver, and rab- 
bit reticulocytes are distinctly different from 
each other. 

Cellular DNA has been found to reside both 
in the nucleus and in mitochondria. Although 
a relatively small portion of cellular DNA is 
found in mitochondria, recent studies have 
shown that in the resting liver cell there is 
a tenfold greater synthesis of mitochondrial 
DNA compared to nuclear NDA, and that mito- 
chondrial DNA has a rapid turnover rate with 
a half life of seven days. Synthesis of nuclear 
DNA is accelerated after partial hepatectomy 
whereas mitochondrial DNA synthesis is un- 
affected. 



Studies of enzymes degrading DNA have 
shown the existence of a group of DNases 
which consist of a mixture of at least 2 endo- 
nucleases and 1 phosphodiesterase. The latter 
enzyme hydrolyzes not only DNA but also 
RNA. 

Work carried out in collaboration with the 
Weissman Institute has revealed that deoxy- 
nucleotides, greater than or equal to 7 in chain 
length, have produced reactivation of the im- 
mune response in thymectomized, X-ray irra- 
diated adult mice. Other work on the biochem- 
istry of immune mechanisms has been pursued 
within the Laboratory of Biochemistry in 
which polylysine models have been examined 
for antigenic capability, that is, ability to pro- 
voke delayed and immediate skin sensitivity. 
Results from these studies suggest that devel- 
opment of delayed responses requires a more 
biochemically complex antigen than that re- 
quired for the development of immediate hy- 
persensitivity, at least as measured in guinea 
pigs. 

A concerted attempt to develop means for 
isolating specific cell types is being directed 
both intramurally and in contract collabora- 
tion with outside facilities. A technique cur- 
rently under exploration is one employing poly- 
urethane foam containing antibody against 
specific cell types to be passed through the 
foam. To date, red blood cells have been con- 
centrated within this system by absorption to 
specific anti-RBC gamma globulin. 

The research activities of the LABORATORY OF 
BIOLOGY, although generally broad in scope, in- 
clude distinct emphasis on work in immunology, 
the behavior of cells under in vitro environ- 
ments, genetics, and carcinogenesis. Research 
in the field of immunology, particularly immu- 
nologic aspects of tumorigenesis and tumor 
recognition, is approached via the plasma cell 
system and the thymus. A thymic humoral 
factor has been identified as being responsbile 
for initiating and maintaining homeostatis of 
the lymphoid cell population controlling immu- 
nologic competence. A new observation is that 
spleen cells from C57BL mice with thymus in- 
tact produce "runt disease" when injected into 
strain A mice because of the graft versus host 
reaction; but if the spleen cells are from a thy- 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mectomized C57BL they lack the immunologic 
competence to induce this reaction. Syngeneic 
thymic grafts in thymectomized C57BL mice 
restore this competence of the spleen cells. 

An inbred strain of mice is being closely ob- 
served because these animals develop, by the 
ages of 12 to 24 months, a reticulum cell neo- 
plasm closely resembling Hodgkin's Disease as 
seen in man. This disease is fatal within a 
1-2 month period after its development. The 
animals carrying the lesions are found to pro- 
duce abnormal immunoglobulins. 

The plasma cell tumor is used in intensive 
studies on the differentiation of the plasma 
cell and its synthesis of specific immunoglob- 
ulins. The functional relationships between 
gene structure and immunoglobulins produced 
indicates that only one gene is operative in pro- 
ducing the heavy chain of the immunoglobu- 
lin molecule and one gene operative in produc- 
ing the light chain; all other possible operative 
genes being repressed during that time. 
Identification of immunoglobulins of mice is 
done by means of chain specific antisera pre- 
pared in inbred strains. 

The genetic patterns of tumor development 
are being studied by biochemical means, the 
initial work identifying urinary proteins of 
the mouse by various chemical detection meth- 
ods. These studies have indicated that some 
urinary proteins are under genetic control. The 
effect of specific genes on the occurrence of 
tumors in mice continues to be studied along 
classical lines established in the Laboratory 
of Biology. The development of mammary 
plaques, a pre-malignant lesion, has been found 
to be dependent not only on the genotype of 
the mouse but also on the strain of mammary 
tumor viruses carried by the animal. 

Much of the current research on the behav- 
ior of cells in in vitro conditions have been 
directed toward the malignant transformation 
process and influences within the tissue cul- 
tural environment which modify this change. 
It has been observed that malignant transfor- 
mation of cells occurs more rapidly in the 
presence of tumor virus (e.g. polyoma). It is, 
however, felt that neoplastic transformation 
may occur in cells free of tumor viruses. Addi- 
tional work on tissue culture systems is heav- 



ily focused on the effects of changes in the 
culture medium on the behavior of cells in gen- 
eral, their growth responses and metabolic 
characteristics. 

The biologic identities of hepatomas induced 
by chemical carcinogens (e.g. carbon tetra- 
chloride and fluorenamine compounds) are 
being studied both by light mircroscopic and 
electron microscopic techniques. The identifica- 
tion of ultrastructural characteristics of var- 
ious tumor cells, as well as cells in tissue cul- 
ture, is only in its early stages of development 
in this laboratory. 

The LABORATORY OF pathology and the 
pathologic anatomy BRANCH, although orga- 
nizationally distinct units, maintain an intimate 
functional relationship. It is, however, recog- 
nized that the primary responsibility of the 
Pathologic Anatomy Branch is to provide diag- 
nostic pathologic service to the several Insti- 
tutes, as well as to pursue research related to 
human disease. On the other hand, the Labora- 
tory of Pathology is involved primarily with 
experimental cancer research. 

The research activities of the Pathology 
units collectively may be grouped into three 
major categories, namely cancer in man, can- 
cer in animals and viral carcinogenesis. A fur- 
ther breakdown reveals a range of research in- 
terests and includes ongoing research in the 
following: biochemistry, carcinogenesis (chemi- 
ical, parasitic, hormonal, viral), viral oncology, 
immunology, comparative oncology and geo- 
graphic pathology, in addition to usual studies 
in human pathology. 

Recent biochemical work on the mechani- 
isms of interferon action have shown that the 
synthesis of interferon is dependent upon pro- 
tein synthesis and is inhibited by such chemi- 
cals as puromycin or p-fluorophenylalanine. 
Interferon in turn blocks synthesis of viral 
DNA and RNA. 

A number of studies are concerned with car- 
cinogenesis in man and animals. Several proj- 
ects are pursued to determine the carcinogenic- 
ity of a large number of chemical agents on 
one or more organ systems in small laboratory 
animals and in sub-human primates. The lat- 
ter animals were used in carcinogenicity stud- 
ies as long as 17 years ago when 3 MCA was 



NATIONAL CANCER INSTITUTE 



instilled in the stomach wall of a group of 
monkeys; no neoplastic lesions have been de- 
tected to date in these animals which are still 
under observation. Currently infant monkeys 
are receiving, either in the diet or sub-q, sev- 
eral carcinogenic chemicals including methyl- 
cholantrene, benzpyrene, aso-dyes, fluorena- 
mine compounds, urethane, aflotoxin, and 
diethyl nitroso amine. The last named chemical 
has induced 13 hepatomas and the tumor has 
been propagated to date via intracerebral 
transplantation in monkeys. Another monkey 
has developed chronic myelocytic leukemia in 
response to MIH, a chemotherapeutic drug 
currently in use in man. The phenomenon of 
vitamin anti-carcinogenesis, mentioned ear- 
lier, has been identified with vitamin A which 
has been shown to prevent cervical cancer and 
stomach cancer in hamsters given dimethyl- 
benzanthracene. 

An important relationship between the state 
of cellular differentiation and susceptibility 
to carcinogenic influences has been illustrated 
by the finding that a certain stage of develop- 
ment of mouse salivary gland is necessary 
before it is susceptible to the tumorigenic influ- 
ences of polyoma virus. The presence of mesen- 
chyme in turn is required for this differentia- 
tion to occur. 

A number of studies continue to explore the 
relationships between several viruses and can- 
cer or cancer-like diseases, and reference is 
made to the review of this work contained in 
the Summary Statement of Pathology. 

A significant observation in the functional 
response of lymphoid cells in tissue culture 
has been made on cells, isolated from a patient 
with lymphoma and maintained under in vitro 
conditions. These cells have been found to syn- 
thesize immunoglobulins. It has not been ascer- 
tained whether these are produced in response 
to a specific antigen, viral or otherwise. 

The research interests of the scientific staff 
of the laboratory of physiology constitute a 
somewhat wide spectrum which would include 
the following subjects: metabolic aspects of can- 
cer, UVL carcinogenesis, thyroid biology and 
physiology, biochemistry (protein, nucleic 
acids), radiation biology, mathematical biology 



and programming, pharmacobiology and im- 
munology. 

Recent, but unpublished, work indicates the 
short ultraviolet wave lengths (2537A) may 
be carcinogenic. The usual carcinogenic wave 
length at the earth's surface is found at 2969A, 
the erythema producing wave length, and no 
VL of 2537A normally reaches the earth's 
surface. Whereas the shorter wave lengths 
may be an additive carcinogenic factor, it 
should be recalled that earlier work in the 
Laboratory of Physiology has shown that longer 
(visible) light is protective against UV car- 
cinogenesis. 

Studies of the biologic behavior of the thy- 
roid gland have revealed that the resorption 
of colloid occurs by a seeming combination of 
mechanical and enzymatic events in which col- 
loid is taken into the peripheral cells of the 
follicles via pseudopodic entrapment by the 
cells; actual resorption of the colloid seems to 
occur intracellular^, presumably by enzymatic 
digestion. Recent studies in this unique organ 
have dealt with the kinetics through which 
the follicles establish and maintain a concen- 
tration of iodide above that of the blood. 

Inhibition of RNA sysnthesis in Sarcoma 
37 cells by actinomycin has been shown to re- 
sult in diminished synthesis of proteins, in- 
cluding histones, and sterols. The inhibitory 
effect is reversed by glucose and other gly- 
colyzable substrates. Another study of intracel- 
lular control mechanisms has involved the in- 
hibition of hemoglobulin synthesis by both 
iron and heme. It is felt that this inhibition 
occurs via effects on polyribosome assembly. 

Mechanisms of recovery from radiation 
damage are being studied from several direc- 
tions. In addition to the phenomenon of "spon- 
taneous" recovery it has been found that cer- 
tain compounds (e.g. colchicine, endotoxin) 
protect cells against radiation damage when 
given after or before exposure. Isolated 
macromolecules in a dry state are found to be 
protected against radiolysis by metallic ions. 
Studies of the possible mechanisms by which 
a cell repairs radiation damage, in which DNA 
synthesis is controlled or inhibited, have indi- 
cated that DNA synthesis is not necessary for 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



repair to occur, whereas RNA synthesis may- 
be required. 

Recovery from radiation affects, and pro- 
tection against effects, are further explored 
in studies on radiation inhibition of natural 
antibody synthesis and recovery thereof, and 
in studies involving the tolerance or rejection 
of allogeneic bone marrow grafts and graft 
versus host reactions. The immunologic behav- 
ior of allogeneic bone marrow in animals has 
been found to be greatly modified by various 
manipulations of the bone marrow cells prior 
to innoculation. It may be wondered whether 
the antigenic identity of the cells is in any 
way changed by the manipulations. 

The Clinical Branches 

The clinical and laboratory research program 
of the dermatology branch has continued 
along lines developed in recent years with the 
addition of two new laboratory activities, 
namely research in cell biology and electron 
microscopy. The major groups of the research 
interests are: (1) Mycosis fungoides lymphoma, 
(2) Epidermal growth and differentiation, (3) 
Biology of lymphoreticular cells, and (4) Mela- 
nogenesis. 

The clinical research program conducted 
during the past year within this Branch has 
almost entirely involved the immunologic re- 
activity of patients with lymphona, particu- 
larly in a comparison of patients with mycosis 
fungoides with Hodgkin's Disease and other 
disease conditions having alterations in im- 
mune responsiveness. Mycosis fungoides re- 
mains as a distinct lymphoma insofar as 
patients with this disorder retain intact immu- 
nologic capabilities. 

An incidental finding has been made in pa- 
tients with mycosis fungoides topically treated 
with HN 2 . Most patients receiving such treat- 
ment have developed cutaneous sensitivity to 
this drug. However, the topical sensitivity 
either disappears or is markedly diminished 
following intravenous administration of HN 2 . 

Reorganization within the past year resulted 
in the transfer of portions of the endocrinol- 
ogy branch to the NICHHD with the result 
that the Endocrinology Branch, NCI, was re- 
duced to approximately half its previous size in 



both physical facilities and personnel. The re- 
search program of the Endocrinology Branch 
has been reoriented toward studies of androgen 
and estrogen synthesis and metabolism. Specific 
laboratory projects, and the recruitment of sci- 
entific personnel to implement the Branch pro- 
gram, have been initiated. 

The Endocrinology Branch has further ex- 
tended its interests in the problem of breast 
cancer. It has initiated explorations into pos- 
sible means to enhance the development of 
knowledge that will lead to a more precise 
definition of the disease and its therapeutic 
control. 

With the recognition that eventual thera- 
peutic control of breast cancer will require 
identification of a number of biologic deter- 
minants operative in the genesis and course of 
the tumor, the Branch places heavy emphasis 
on studies of hormonal patterns that may be 
associated with breast cancer, the isolation of 
prolactin and its quantitative chemical assay, 
and biologic assay of prolactin as measured 
by its effects on the mouse mammary gland. 
The latter at this time is planned to be initi- 
ated by collaborative involvement of other re- 
search units within and outside of NCI. 

The immunology branch functions as the 
focal organizational unit within the GL&C en- 
tirely concerned with immunologic aspects of 
the cancer process. In addition to its own inten- 
sive research activities, it has provided labora- 
tory facilities and guidance for the development 
of immunologic research in other branches and 
for further training of scientific personnel in 
immunologic techniques. 

Evaluation of human sera for immunoglob- 
ulin constituency has been initiated through a 
contract with a commercial laboratory (Mel- 
par). Through this means it is planned that a 
large number of sera, representing a wide range 
of disease states, will be so evaluated. The re- 
sults will be reviewed for possibilities of iden- 
tifying immunodiagnostic criteria. 

Lymphoid cells from a human lymphoma 
have been established in continuous culture. In 
collaboration with scientists in the Pathologic 
Anatomy Branch, these cells have been found 
to produce immunoglobulins and constitute 



NATIONAL CANCER INSTITUTE 



the first possible model to study the produc- 
tion of immunoglobulins by lymphoid cells in 
in vitro conditions, with the goal of producing 
specific human antibody. 

Further research activities of this Branch 
are identified in the special review entitled 
"Evaluation of Immunologic Research in 
GL&C," a separate item in this report. 

The metabolism BRANCH primarily conducts 
research relating either secondarily to the can- 
cer process or to matters frequently directly 
consequent to cancer, e.g., anemia of cancer. 
Research currently active includes the follow- 
ing: nucleic acid metabolism, amino acid trans- 
port, metabolism of albumin and globulin, por- 
phyrin metabolism, erythrophoresis, calcium 
metabolism and hemaglobulin synthesis. Refer- 
ence is made to the review given these studies 
in the summary report of the Branch. 

Selected attention is drawn to the findings 
during the past year from studies on porphy- 
rin metabolism. Delta-aminolevulinic acid syn- 
thetase (ALA synthetase) has been partially 
purified from mitochondria. The kinetics of 
induction of hepatic ALA synthetase have 
been studied along with the effects of inhibi- 
tors of protein synthesis (puromycin, actino- 
mycin-D, 5-fluorouracil, p-fluorophenylalan- 
ine, etc.). Glucose blocks induction of this 
enzyme as effectively as actincmycin-D. It has 
been shown that heme, the end product of the 
pathway which ALA synthetase controls, will 
prevent induction of ALA synthetase when in- 
jected intravenously at certain times before 
induction is initiated. This is the first direct 
evidence of heme as a repressor of ALA syn- 
thetase in mammalian tissues and supplements 
the indirect evidence from the tryptophane 
pyrrolase work reported last year. When heme 
is injected intravenously, the level of hepatic 
ALA synthetase oscillates markedly for periods 
of 4 to 5 days after a single dose. The oscilla- 
tions can be greatly amplified by administra- 
tion of inducer 5 hours before sacrifice. This 
probably is the first demonstrated example of 
significant oscillations of the level of an en- 
zyme. The only other example is reduced nico- 
tinamide adenine dinucleotide oscillations in 
in vitro yeast extracts of glycolyzing systems. 
The oscillations of ALA synthetase in mammal- 



ian liver explain Watson's observations on var- 
iations in bilirubin excretion following heme 
administration and raise the question of the 
role of ALA synthetase in biologic clock mech- 
anisms. The effects of diet on preventing the 
chemical abnormalities of acute porphrin pre- 
viously demonstrated in the experimental ani- 
mal have been extended to patients. A de- 
creased caloric intake can initiate attacks of 
the disease and high carbohydrate diets can 
end these attacks. A small fraction of women 
whose attacks of acture porphyria recurred 
cyclically with their menstrual periods have 
been maintained free of disease by use of oral 
anovulatory hormone preparations. 

DNA-RNA hybridization techniques have 
been used to study the taxonomy of microor- 
ganisms, mycoplasma and streptococci. Myco- 
plasms have been found to represent a highly 
heterogeneous group of organisms by this 
technique. The reliability of the hybridization 
technique for the identification of microorgan- 
ism types is supported by the fact that the 
results correlate exceedingly well with those 
from usual serologic typing of these organisms. 

A major responsibility of the surgery 
branch is to provide consultative advice and 
service to the several Institutes in clinical prob- 
lems requiring judgment in general, urological, 
otolaryngological, gynecological or thoracic sur- 
gery. In addition to this, a clinical and labora- 
tory investigative program, broad in scope, is 
actively carried out. Much of the research is 
directly concerned with development and eval- 
uation of surgical procedures applied to control 
of cancer in man, and control of complications 
secondary to cancer. Examples include clinical 
evaluation of combined ionizing radiation and 
surgery as a cancer therapeutic modality; and 
evaluation of pre- and post-operative prophy- 
lactic antibiotic administration to control infec- 
tious complications of cancer surgery. A study 
of the latter has shown that chloramphenicol, 
given pre- and pcst-operatively over a ten-day 
period, has markedly diminshed infectious com- 
plications to the low frequency of 7 % compared 
to 22-30% in patients receiving no antibiotic 
therapy. 

The Surgery Branch has been provided two 
laser units with which exploration of the ap- 



8 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



plicability of this type of radiation to research 
and treatment of disease in man and animals 
has been initiated. Pursuit of investigations 
utilizing these instruments will be in accord 
with initial results obtained and the likelihood 
of deriving significant information with their 
continued use. 

Greater and greater emphasis is placed on 
research on immunologic processes involved in 
transplantation, cellular recognition, and con- 
trol of the cancer process. Several studies are 
in progress. A study of the immunologic rela- 
tionship between pregnant women, or women 
with a trophoblastic tumor, and their hus- 
bands has been carried out using the technique 
of lymphocyte transformation in mixed leuko- 
cyte cultures. Preliminary results show that 
lymphocytes from normally pregnant women 
do not transform when grown with their hus- 
band's leukocytes in as high a percentage as 
they do when grown with cells from an unre- 
lated male. Further studies are being carried 
out to evaluate serial changes in each trimester 
of pregnancy and the changes that take place 
during and after chemotherapy of tropho- 
blastic neoplasms. 

A chemically-induced gastric adenocarcinoma 
has been found to be antigenic eliciting an 
'immunologic response which can be adop- 
tively transferred by isologous lymphoid cells, 
and which may be abrogated by splenectomy 
or large doses of tumor cells. Attempts to uti- 
lize sensitized lymphoid cells to alter the 
growth of established tumors have resulted in 
possible slowing of tumor growth; complete 
regression has not occurred. 

Immunologic Research 

As earlier pointed out, while the research in- 
terests and activities within the GL&C, NCI, 
extend over a broad range of subject inter- 
ests, there are several subjects in which there 
is an intensity of interest and work. These 
include biochemistry, carcinogenesis, virology 
and immunology. Because of increasing evi- 
dence of the importance of immunologic factors 
in the development of some neoplasms, and 
an awareness of possible applicability of im- 
munologic factors to control the course of neo- 



plasms, a summary assessment of immunologic 
research within the GL&C seems appropriate. 

A significant amount of research directly 
related to or involving cancer immunology is 
found in the Laboratories of Biology and Bio- 
chemistry; and in the Immunology, Surgery, 
Pathologic Anatomy, Metabolism and Derma- 
tology Branches. 

Immunologic research has been fostered and 
intensively pursued in the Laboratory of Biol- 
ogy for a number of years. Currently the work 
within this Laboratory is directed towad elicit- 
ing antigenic identities of cancer cells; deter- 
mining the rcle exerted by thymic and 
lymphoid cells in directing immunologic capa- 
bilities of the organism; and determining molec- 
ular specificities of immunoglobulins synthe- 
sized by the plasma cell. To date it has been 
shown that some cancers of animals, particu- 
larly viral cancers, have cell-related antigenic 
characteristics which can be utilized to pre- 
vent or modify the early development or course 
of these tumors. Lymphoid cells of thymic or- 
igin have been shown to be highly functional 
in determining and maintaining immunologic 
competence of the mouse. A number of im- 
munoglobulins, each molecularly specific in 
composition, have been identified as products of 
plasma cell tumors. It is found that each tumor 
continues to synthesize a specific globulin. The 
specificity of the stimulus accounting for this 
is under study. In this experimental system, 
no antibody function of the immunoglobulins 
has been discerned as yet. 

Some studies of molecular identities of im- 
munoglobulins produced by plasma cell tumors, 
and factors controlling their synthesis, are 
pursued within the Laboratory of Biochemistry 
in collaboration with scientists of the Labora- 
tory of Biology. Additional research within 
the Laboratory of Biochemistry on the chemi- 
cal basis of immunity has been concerned with 
molecular determinants of antigenicity. Re- 
sults to date, from studies utilizing polylysine 
models, indicate the induction of delayed hy- 
persensitivity requires an antigen with struc- 
ture more complex than that required to in- 
duce immediate hypersensitivity. 

Research of the Immunology Branch is pri- 
marily directed toward studies in man. Scien- 



NATIONAL CANCER INSTITUTE 



9 



tists within this branch have over the years 
provided leadership in research which has led 
to better understanding of the biochemical 
identities and specificities of immuno-proteins 
in man. Work in animals has yielded funda- 
mental information on the reactivities of the 
various components of complement and the 
applicability of complement fixation tests to 
determine antigenic identities of chemically- 
induced tumors. The screening analysis of hu- 
man sera now underway in the Branch is di- 
rected toward identification of immunoprotein 
patterns which might be diagnostic of certain 
types of cancer in man. The work initiated 
collaboratively with scientists of the Pathologic 
Anatomy Branch has provided the first in- 
stance of continual immunoglobulin synthesis 
by lymphoid cells in tissue culture. Inasmuch 
as these cells are of human origin, the possi- 
bility is now raised of in vitro synthesis in 
other culture systems of specific antibody for 
human use. 

Immunofiuorescent studies of possible tumor 
specific antigens in man and animals have been 
carried out in the Pathologic Anatomy Branch 
in recent years. Some of these studies have 
suggested that specific tumor antigens may 
exist in man, although a definite conclusion 
cannot be made at this time. Collaborative 
work along these lines is being continued with 
other units in GL&C and with units in the 
other program areas of NCI. 

Immunologic research of the Surgery 
Branch has to date yielded positive informa- 
tion on the role of the small lymphocyte in 
maintaining immunologic competence in man. 
Partial depletion of the lymphocyte population 
by thoracic duct drainage has resulted in sig- 
nificant diminishment of delayed immune re- 
activity as measured by skin tests and homo- 
grafts. Immunologic research capabilities are 
being actively encouraged within this branch. 

Studies of the Dermatology Branch have 
shown that the patients with mycosis fun- 
goides lymphoma remain immunologically com- 
petent throughout the disease, in distinction 
to patients with Hodgkin's Disease. Immuno- 
logic challenges, cutaneous and systemic, of pa- 
tients with mycosis fungoides have been ob- 



served to effect partial or complete remissions 
of the disease in some patients. 

Results of clinical and laboratory work to 
date suggest there is a likelihood of influenc- 
ing cancer in man by immunologic means. It 
is intended that early leads will be tested as 
quickly as possible in man, but at a pace and 
in a fashion commensurate with standards ap- 
propriate to research in man. 

MACROMOLECULAR BIOLOGY SECTION 

In the recent reorganization of the NCI, the 
Macromolecular Biology Section was retained 
in the intramural research area in the Office 
of the Scientific Director for General Labora- 
tories and Clinics, NCI. The Macromolecular 
Biology Section was previously under the 
Laboratory of Viral Oncology, which has been 
transferred to Field Studies, Etiology. The de- 
cision was made to retain the Macromolecular 
Biology Section in the general intramural re- 
search area since the basic research in the 
Macromolecular Biology Section pertains to 
cell growth and differentiation in general, as 
well as to viral oncogenesis. 

During the past few years there has been 
increasing recognition that the new techniques 
and findings of molecular biology may lead to 
significant clarification of cellular differentia- 
tion on the macromolecular level. Differential 
macromolecular synthesis and differential uti- 
lization of energy through processes controlled 
by macromolecules are crucial in the control 
mechanisms of histo-differentiation. In order 
to understand the control mechanisms of can- 
cerous transformation, greater understanding 
must be gained of the processes of differentia- 
tion, cell division, and contact inhibition. 

The combined disciplines of molecular biol- 
ogy (biophysics, genetics, virology, biochem- 
istry, polymer and physical chemistry) were 
fused in recent years into a unique research 
facility at NIH in the Section of Macromolec- 
ular Biology. The aim was exploration of the 
role of macromolecules in cellular control 
mechanisms. Thus the research emphasized the 
interaction of macromolecules in the cellular 
control processes which govern differential 
synthesis of macromolecules, utilization of en- 
ergy, and growth of cells. 



10 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



In the field of macromolecular interactions 
and protein structure pertaining to control 
processes in energy utilization, Dr. Mora, with 
associates, was studying the changes in cyto- 
chrome c structure associated with intercon- 
version between the oxidized and reduced 
states. Certain polycations, prepared by substi- 
tuting polyglucose with basic residues, and 
certain sugars, reduce cytochrome c in a 
unique, slow and controllable fashion, so that 
both the rate and the extent of reduction of 
the cytochrome c can be varied. This technique 
permits observations on the changes in the 
tertiary structure of this respiratory protein 
during its conversion from oxidized to reduced 
form, heretofore impossible. Greater under- 
standing of protein conformational changes 
in cytochrome c allows better understanding 
of the control processes of oxidative phos- 
phorylation in terminal electron transport. Cy- 
tochrome c plays a central role in terminal 
electron transport in the mitochondria and in 
energy utilization in all types of cells. 

Dr. Shifrin shifted his research interest to 
the study of cellular control processes in bac- 
teria. In collaboration with investigators in 
the Laboratory of Chemical Biology, NIAMD, 
he studied the aggregation of the subunits of 
/?-galactosidase isolated from wild-type E. 
coli and from a mutant which lacks /?-galac- 
tosidase activity. He found that a tetramer of 
the subunit is required for the enzymatic func- 
tion, and a point mutation in the lac operon 
yields a protein which is enzymatically inac- 
tive primarily because the site required for ag- 
gregation of the subunits of the /?-galactosi- 
dase to an active tetramer is mutated. In an- 
other collaborative work with scientists in the 
Laboratory of Molecular Biology, NIAMD, Dr. 
Shifrin studied the mechanisms of action of 
an amino acid analogue, the a-hydrazino ana- 
logue of histidine, which inhibits bacterial 
growth. The study utilized a mutant of S. ty- 
phimurium defective in histidine transport 
system. He found that this metabolic inhibitor 
is functioning not by virtue of its ability to 
be incorporated into the protein and thus make 
a defective enzyme, but by acting as a feed- 
back inhibitor. The site of action was found to 
be on the acetylornithine transaminase in the 



arginine biosynthetic pathway. Thus, an im- 
portant linkage between the bacterial cell wall 
or membrane where materials are transported, 
and the control of cellular growth is clearly 
demonstrated in this study. 

Other work pertained to macromolecular 
differences in higher organisms. In one study, 
Dr. Smith carefully compared the nucleic acids 
of rat liver and hepatoma from the point of 
view of base sequence homology. Refined and 
novel physical measurements were used. Ob- 
servation of the changes (upon heating) in 
the optical properties of the DNA at various 
wavelengths, and comparison of the so-called 
"melting" profiles revealed no difference in 
the DNA from the two tissues. In addition, 
various techniques were employed in hybridiz- 
ing denatured single-stranded nucleic acids 
from the two tissues. Currently attempts are 
being made to compare liver and hepatoma 
messenger RNA by various hybridization tech- 
niques designed to reveal differences in their 
base sequences. 

The study of differences in macromolecular 
pathways of normal cells and tumor cells can 
be conveniently accomplished by the compari- 
son of normal cells and tumor virus-trans- 
formed cells in tissue culture. Preliminary puri- 
fication and characterization of the virus and 
its nucleic acid are essential. 

With the help of a small contract at Mel- 
par, Inc. (which expands the limited tissue 
culture facilities of the MBS), Drs. Luborsky 
and Wood studied the incorporation of tritium 
into DNA in mouse cells infected by the poly- 
oma virus. Under specific conditions, in the 
presence of FUDR and tritiated thymidine, it 
was found that only viral DNA was synthe- 
sized in mouse embryo cells. In the absence of a 
very critical concentration of the metabolic 
inhibitor the major amountt of the newly syn- 
thesized DNA is induced cellular DNA. These 
findings were possible after first developing a 
fractionating technique so that the cellular 
DNA was fully separated from the viral DNA 
on methylated albumin column, and after 
studying the balance of the synthesis of these 
two DNA species in the cells under various con- 
ditions. Conditions were also found to in- 
crease the level of incorporation of the tritium 



NATIONAL CANCER INSTITUTE 



11 



into the virus DNA. The highly radioactive 
viral DNA is used currently in refined nucleic 
acid homology studies, following the pioneer 
observations of Axelrod, Habel and Bolton. The 
objective is to see what part of the viral ge- 
nome is incorporated into the transformed 
tumor cells in different types of polyoma- 
induced tumors. Another objective is to see if 
the viral DNA genome, or a fragment, incor- 
porates perferentially into the transformed 
cell's mitochondrial DNA or into its nuclear 
DNA. Further objectives pertain to changes 
in macromolecular synthetic pathways and to 
cell surface changes during transformation of 
cells to cancer cells (see below). 

A significant achievement in the Macramo- 
lecular Biology Section was the first isolation 
of an intact RNA from an animal leukemia 
virus. A 13 million molecular weight single- 
stranded RNA molecule was isolated from the 
Rauscher mouse leukemia virus. It was shown 
that this molecule accounts for the full genome 
of the virus. To our best knowledge this RNA 
is the highest molecular weight RNA ever ob- 
served. Our method of isolating it in good yield 
opens the way for molecular biological studies 
of the RNA-containing viruses. One important 
finding in this respect is that the RNA is sin- 
gle-stranded. Questions which are now under 
study include: whether and how transforma- 
tion or virus induction may occur with a pure 
and intact single-stranded viral RNA; does 
this RNA possess the full genetic information 
for transformation, or is other information 
necessary? 

A particularly important change during 
transformation of a cell from the normal to 
the malignant state must be on the cell sur- 
face regulating the growth of the cell vis-a-vis 
the other cells. Acid mucopolysaccharides have 
been reported to be qualitatively different after 
virus infection and transformation of several 
cellular systems. Chemical methods are being 
developed in the Macromolecular Biology Sec- 
tion for the isolation and characterization of 
these mucopolysaccharides from various nor- 
mal cells and from virus producing and virus 
transformed cells. (Dr. Wood) 

The possible changes in surface charge dur- 
ing transformation of cells, and the effect of 



added polyanions and polycations on cell 
growth and on contact inhibition of normal 
and malignant cells are also being studied in 
tissue culture. (Dr. Schutz) Techniques for 
measuring DNA synthesis, rate of mitosis, 
cell overgrowth, etc., is being refined to per- 
mit reproducible measurement of the changes 
induced in cell growth by charged polymers. 

Lipid components of normal cells and of cells 
producing certain viruses, especially the so- 
called membrane viruses were compared (Dr. 
Johnson). It was found that more unsaturated 
and short chain lipids were produced in cells 
which were infected by the Rauscher, Moloney 
or Rous viruses, as well as in Rous-transformed 
cells. 

Future research will continue to center 
around the molecular basis of differentiation. 
With the help of two additional investigators, 
research on protein biosynthesis in subcellular 
systems and electron microscopic investiga- 
tions on cell surface changes will be integrated 
into this effort. 

LABORATORY OF BIOCHEMISTRY 

Cytochemistry Section 

Dr. D. Burk and Dr. M. Woods have con- 
tinued their investigations of the effects and 
modes of action of anti-cancer agents on the 
metabolism, growth, and death of cancer cells. 

Cancer tissues and cells have been exposed 
in vitro and in vivo to chemical or physical 
(hyperthermy, laser, laser, light) agents, and 
effects on their metabolism, growth, and death 
have been studied. In vitro exposures of 30-90 
minutes to temperatures of no more than 43- 
44° C sufficed to produce extensive or total 
killing of animal cancer cells, results similar to 
those obtained clinically by von Ardenne and 
Kirsch in Dresden, Germany. The thermal 
death temperatures and exposure times in- 
volved varied somewhat with the cancer cell 
type, and were lowered by certain drugs, such 
as steroids and serotonin, and raised by insulin 
and glucose. In vivo exposures to water tem- 
peratures of 43-44° C (90% total body im- 
mersion of host mouse) indicate that the tu- 
mor cells are more readily killed under in vivo 
than in vitro conditions, due in part to im- 



12 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pairment of the tumor blood flow and subse- 
quent decreased glucose supply and anoxia. 
Concommitant with loss of viability as meas- 
ured by transplantation is a greatly decreased 
respiratory capacity and Pasteur Effect (inhi- 
bition of fermentation by oxygen gas), fol- 
lowed later by greatly decreased fermentation 
capacity. The in vitro metabolism and in vivo 
growth of two different tissue culture sarcoma 
cells lines, both derived years before from the 
same single cell, have remained relatively con- 
stant to each other over the last eight years 
in tissue culture. 

Drs. Burk and Woods are the recipients of 
the 1965 Gerhard Domagk Prize for Cancer 
Research which was awarded for their paper 
showing the connection between glucose fer- 
mentation and growth rate in the spectrum of 
Morris hepatomas. 

Dr. W. H. Evans in his biochemical studies of 
normal and leukemic leukocytes has joined 
with Dr. M. Rechcigl, Jr. in an examination 
of the peroxidative metabolism of leukocytes. 
Little is known about the relative roles of cata- 
lase and peroxidase although elevated catalase 
activity of leukocytes has been reported in 
chronic myelogenous leukemia. 

The present studies indicate that myeloper- 
oxidase in the intact cell is active in the pres- 
ence of sufficient aminotriazole to inhibit cata- 
lase. With cells disrupted by homogenization, 
however, both enzymes are inhibited. New data 
indicate that the inhibition of myeloperoxi- 
dase by amiotriazole depends on the presence 
of a peroxide-generating system whereas cata- 
lase can be inhibited by amiotriazole but un- 
der conditions where a much lower peroxide 
concentration exists. Inhibition by aminotria- 
zole of both myeloperoxidase and catalase also 
occurs when leukocytes are caused by phago- 
cytize polystyrene particles, another condition 
leading to increased hydrogen peroxide for- 
mation in intact cells. These observations sug- 
gest that catalase and myeloperoxidase are lo- 
cated in different compartments within the 
cell. Purified myeloperoxidase has been shown 
to oxidize formate-C 14 to C 14 2 in the pres- 
ence of H 2 2 . Myeloperoxidase may account 
for the major part of formate-C 14 oxidation 
in leukocytes since catalase activity can ac- 



count for only about 30% of the peroxidative 
metabolism of leukocytes as measured by for- 
mate-C 14 oxidation. 

Nutrition and Carcinogenesis Section 

Transplantable hepatomas, originally de- 
rived by Dr. H. P. Morris from primary hepa- 
tomas induced by a variety of aromatic 
amines, provide a spectrum of tumors which 
are being used to examine the relationship be- 
tween biochemical and biological characteris- 
tics and degree of malignancy. A total of 24 
cooperating laboratories in the United States 
and abroad are involved in this continuing 
study. The growth rate of these transplantable 
tumors differs many fold and, morphologically, 
they vary from poorly or undifferentiated to 
highly differentiated neoplasms. 

Many tumors with biochemical characteris- 
tics similar to those of normal liver are slow 
growing, have reduced tissue-specific antigens, 
are not completely devoid of the slow-moving, 
h 2 electrophoretic class of proteins, and have 
a reduced capacity to incorporate thymidine 
into DNA. A high correlation has been estab- 
lished in cell free systems between growth 
rates of these hepatomas and the activities of 
the replicative and terminal DNA nucleotidyl 
transferases, the faster growing tumors hav- 
ing the higher activities. Degradative enzymes 
and kinases in such cell free systems were not 
rate-limiting. The very slow growing hepato- 
mas appear to have more organized endoplas- 
mic reticulum and fewer free ribosomes. Some 
of the free polyribosomes will bind to mem- 
branes from normal liver but polyribosomes, 
originally bound to membranes of normal rat 
liver, do not bind to membranes from hepa- 
tomas. 

It has been amply demonstrated that this 
spectrum of hepatomas provide a series with 
graded biochemical alterations, with some bio- 
chemical parameters increasing with growth 
rate, others decreasing, while still others re- 
main unchanged. Biochemical characterization 
seems to be much more specific than morpho- 
logical characterization since, despite the large 
number of tumors with different growth rates 
but similar morphology that have been de v el- 



NATIONAL CANCER INSTITUTE 



13 



oped, hepatomas with identical biochemical 
patterns have not been noted. 

Glutamic-oxalacetic transaminase (GOT) 
activity in these transplantable rat hepatomas 
has been shown by Drs. T. T. Otani and H. P. 
Morris to consist of two isozymes. Studies of 
the distribution of these isozymes, their 
Michaelis constants, and the effect of hor- 
mones have been made in the hepatoma and 
the liver of tumor-bearing animals. The host 
liver GOT content and isozyme distribution 
was not affected by adrenalectomy, hydrocor- 
tisone treatment, a combination of both nor 
by the presence of tumor. With the 5123B 
hepatoma, however, while hydrocortisone 
treatment or adenalectomy alone showed no 
effect, there was a shift in the isozyme dis- 
tribution of the tumor in females, but not in 
males, when hydrocortisone was administered 
to an adenalectomized animal. 

Dr. Morris has also been studying the role 
of tryptophan and indole in the induction of 
bladder tumors in rats ingesting N-2-fluorenyl- 
acetamide (2-FAA) and the effects of the level 
of 2-FAA on the induction of tumors in jaun- 
diced rats. Although all details of these experi- 
ments have not been completed, as reported 
last year, it appears that bladder tumors can 
be produced in Fischer strain rats without the 
addition of tryptophan. Jaundiced rats ingest- 
ing 0.025% 2-FAA develop as many liver tu- 
mors as do non jaundiced animals of the same 
genetic background. Lower levels of 2-FAA are 
now being studied to determine whether the 
early results were due to an overwhelming ex- 
posure to the carcinogen. Animal experiments 
have been completed and histologic studies are 
in progress. 

A series of "unnatural" /?-hydroxyamino 
acids and their derivatives have been synthe- 
sized by Dr. T. T. Otani for use as growth in- 
hibitors and racemic mixtures of disasterom- 
ers were tested. Certain of these were shown 
to possess moderate growth inhibiting activity 
in microbial and solid tumor tests systems. 
Since only one out of the four diasteromers is 
presumed to be active, increased inhibitory 
activity should result from the use of the pure 
resolved isomer. Milligram quantities of each 
of the four isomers of /?-hydroxynorleucine 



and their N-chloroacetyl derivatives have been 
prepared and remain to be tested. 

Tumor-Host Relations Section 

The study of the in vivo environment of the 
neoplastic cell by Dr. P. Gullino has been di- 
rected toward an examination of the energy 
requirements of growing tumor tissue. By use 
of the "tissue isolated" tumor preparation into 
which has been implanted a chamber for har- 
vesting interstitial fluid, blood flow, glucose, 
and lactic acid levels, and 2 and C0 2 ex- 
change levels can be measured continuously 
in interstitial fluid and in efferent and affer- 
ent blood. 

In three solid tumors, chosen because of dif- 
ferences in structure and biological behavior, 
it was found that about 50% of the available 
blood glucose was removed from the blood and 
utilized in different degree by each tumor. The 
limiting factor in oxygen utilization seems to 
be the supply since, within the limits of the 
50% removal ratio, the three tumors used all 
the 2 that was received. It was not possible 
to saturate the ability of the neoplastic tissue 
to use 2 . Conversely, neoplastic tissues appar- 
ently were unable to remove a larger propor- 
tion of 2 from the blood when less is supplied. 
A decrease of arterial 2 from 20 to 5 vol- 
umes percent resulted in a decrease of 2 used. 
Furthermore, during acute in vivo lack of 2 , 
the tumors failed to respond with increased 
glucose utilization of lactate production. Glu- 
cose utilization and lactate production were di- 
rectly related to 2 consumption; no "spar- 
ing" effect (Pasteur effect) of 2 on glucose 
utilization could be demonstrated in vivo. 

In normoglycemic animals a sharp gradient 
of glucose concentration was found between 
plasma and interstitial fluid (from 150 mg/ 
100 ml plasma to 0-8 mg/100 ml interstitial 
fluid). The tumors utilized all the glucose which 
passed through the capillary wall. The gradient 
was lost in hyperglycemia and restored by nor- 
moglycemia. Several hours were needed for the 
two phenomena to occur, suggesting that a 
glucose transfer system is operating across the 
capillary wall of tumors. Hyperglycemia (350 
mg/100 ml plasma) lasting several days in- 
creased glucose utilization to a maximum level 



14 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of about 30% above normal. Higher blood glu- 
cose levels did not induce greater glucose utili- 
zation. Tumors utilizing glucose at the highest 
possible level did not grow any faster; indeed, 
growth was poor in many of them. During nor- 
moglycemia the 3 tumors transformed about 
35% of the glucose utilized into lactate. Dur- 
ing hyperglycemia more glucose was utilized 
and more lactate was eliminated by the 35% 
proportion was maintained. However, in tu- 
mors starved of glucose by severe hypogly- 
cemia (30 mg/100 ml plasma), the amount of 
lactate eliminated increased sharply in propor- 
tion to the amount of glucose utilized. More- 
over, in hyperglycemia produced immediately 
after glucose starvation, the elimination of lac- 
tate in tumors was decreased to negligible 
amounts; some even utilized lactate from the 
afferent blood. These observations imply the 
existence, in vivo, of several compartments in 
the neoplastic cell and that only in some of 
them does glycolysis occur with a special pri- 
ority. 

In an attempt to investigate the relation- 
ship between differentiation and the growth 
of neoplastic cell populations, Dr. P. Gullino and 
Dr. S. Roskes (NICHHD) have begun to em- 
ploy primary mammary tumors in rats. Pre- 
liminary results have shown that the majority 
of primary tumors from lactating hosts have 
a negligible capacity to produce lactose al- 
though it is known that mammary tumors pos- 
sess the enzymes involved in lactose synthesis 
and an appreciable level of activity can be 
demonstrated in vitro. 

It is Dr. E. Shelton's purpose to examine 
the normal and malignant transformation of 
cells. The free cells found in the peritoneal fluid 
of the mouse represent the "wandering cells" 
of the tissue spaces of the body (i.e., the 
extra- vascular wandering cells). These cells 
function by protecting the mouse from adverse 
effects of foreign materials or debris resulting 
from metabolism or death of host cells. In per- 
forming this function, the cells undergo 
changes in number and metabolism. However, 
little is known about the normal development 
of this cell population which must serve as the 
basis for a comparison of malignant cell be- 
havior. 



A study of the population dynamics of nor- 
mal free peritoneal cells has shown that the 
total number of peritoneal cells in the mouse 
rises from birth to a stable plateau at 4 months 
of age. The macrophage population reaches 
a stable plateau of between 2-3 x 10 6 cells at 
30 days. The continued increase in total cells 
is due to the continued increase in total lym- 
phocytes. It is suggested that the increase in 
number of lymphocytes may be related to 
maturation of the immune system in the 
mouse. Major emphasis will be placed upon elu- 
cidating the fine structure of the peritoneal 
cells with reference to the age of the animal. 
Attention will then be turned to the study of 
the alterations in fine structure which are at- 
tendant upon growth in the diffusion chamber. 

As an example of another differentiating cell 
system, whole thymuses from new-born mice 
were grown in diffusion chambers implanted 
in thymectomized or intact, syngeneic or allo- 
geneic hosts of various ages. Whole mount 
preparations were made of the thymus tissue 
after it had grown for from 9-86 days. Thy- 
mic lymphocytes, granulocytes, macrophages 
and fibroblasts migrated rapidly from the ex- 
plant, the latter forming a sheet that covered 
the entire chamber. Viable lymphocytes were 
present in chambers removed from the host 
after 86 days. The striking feature of the cul- 
tures was the differentiation of the epithelium 
into sheets, clusters, and cords of cells remi- 
niscent of glandular epithelium. Whorls of cells 
resembling Hassall's bodies were occasionally 
seen. These epithelial cells were distinguished 
by the presence of large lipid-filled vacuoles 
and neutral mucopolysaccharide in the cyto- 
plasm, suggesting that the epithelial cells were 
engaged in secretory activity. 

In collaboration with Drs. E. L. Kuff and 
W. C. Hymer, Dr. Sheton has examined the fine 
structure of the free and bound ribosomes of 
mammalian cells, principally the RPC 20 
plasma cell tumor of the mouse. Utilizing neg- 
ative and positive staining, the structure of 
these organelles has been visualized by electron 
microscopy in greater detail than hitherto pos- 
sible. The individual mammalian ribosome 
appears as a dual structure consisting of a 
sphere approximately 220 A in diameter 



NATIONAL CANCER INSTITUTE 



15 



(probably corresponding to a 50S ribosomal 
subunit) to which is attached a smaller struc- 
ture of variable morphology (probably corre- 
sponding to a 30S ribosomal subunit). The 
large 50S subunit is extremely stable but the 
morphology of the smaller subunit is variable. 
It has been seen as a flattened sphere, a cleft 
sphere, two small closely apposed spheres and 
various other shapes. The small subunit seems 
to be structurally unstable and instability may 
be related to the capacity of the smaller sub- 
unit to "accept" messenger RNA. Ribosomes 
are attached to membranes of the endoplasmic 
reticulum (ER) by the stable, 50S subunit. 
The unstable 30S subunit stands up and away 
from the membrane. The base of the 50S sub- 
unit appears to form an integral part of the 
ER membrane. 

Polyribosomes are formed by the joining 
together of individual ribosomes into rosettes 
or linear arrays. The patterns formed by the 
rosettes and linear polyribosomes as they set- 
tle out of suspension onto the membrane of the 
microscope grid have provided clues as to the 
way they are joined together. The evidence is 
very strong that the units of the polyribosome 
are joined together at the smaller subunit and 
that the polyribosome strand is flexible. Posi- 
tive stains of the polyribosomes provide evi- 
dence that the material holding the ribosomes 
together is a more substantial substance than 
the single strand of messenger RNA that has 
been postulated previously. 

Dr. M. Rechcigl, Jr. has embarked on stud- 
ies concerned with the selection and differen- 
tiation of cell populations of normal and 
tumor tissues. Understanding the interrelation- 
ships between cells requires systems with mini- 
mum heterogeneity of cell types. At present it 
is not known whether tumor preparations are 
mixed populations of tumor cells and whether 
variation in such populations influence results 
and interpretations by virtue of their varied 
biochemical characteristics. 

It had previously been shown (NCI-388, 
1965) that the injection of pieces of tumor 
into the spleen of a compatible host gives rise 
to numerous metastases in the liver of the host. 
This technique has now been successfully used 
for separation of different cell lines. When 



pieces of tumor from HC-hepatomas (high 
catalase line) were injected into the spleen of 
OM/N rats, all liver metastases found showed 
high catalase activity (i.e., around 200 units). 
Pieces from the low line (LC hepatoma) had 
liver metastases with low catalase values (i.e., 
around 40 units). Freshly prepared mixtures 
from HC- and LC-tumor lines injected into 
spleen produced liver nodules possessing either 
low or high enzymatic activity. No intermedi- 
ate values were seen. Upon reinoculation into 
spleen, the nodules gave rise to liver metas- 
tases of either high or low activity, compar- 
able to the activity of the initial inoculated 
tissue. Similar results were obtained in stud- 
ies with mixtures of unrelated tumors and 
grown in different inbred or hybrid strains. 
Small pieces of a given liver nodule obtained 
from Fj hybrid experiments, transplanted sub- 
cutaneously into both original parent strains, 
would develop tumors only in one of the 
strains. Moreover, the developed tumors pos- 
sessed typical biochemical characteristics of 
the original tumors grown in these strains. 
All the evidence obtained so far implicates 
cloning as the selective process. 

On the other hand, it was established by 
Dr. Kuff that the small tumor nodules observed 
on the mesentary of mice early after intraper- 
itoneal transplantation of ascites plasma cell 
tumors (see NCI-366, 1965) do not exclusively 
represent clones derived from individual tu- 
mor cells. Transplantation of individual nodules, 
while feasible, does not presently appear to be 
a useful cloning technique for these tumors. 

Current studies by Dr. E. L. Kuff and coll- 
aborators are based upon the hypothesis that 
important regulatory mechanisms involved in 
cell reduplication and differentiation may 
have their basis not only in the molecular 
structure of the polyribosomal protein-syn- 
thetic units but also in the relationship of 
these units to the membranes of the endo- 
plasmic reticulum. Transplantable plasma-cell 
tumors, combining cellular proliferation with 
active synthesis of secretory protein, are par- 
ticularly suitable systems in which to study the 
functional activity of intracellular compo- 
nents. 



16 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Cytoplasmic extracts of the RPC-20 plasma 
cell tumor, fractionated by sucrose density 
gradient centrifugation yield 4 major frac- 
tions: (a) a "membrane" fraction containing 
both microsomes with associated ribosomes 
and mitochondria, (b) membrane-free poly- 
ribosomes; (c) free monomeric ribosomes, and 
(d) soluble fraction. The particulate fractions 
have been examined by electron microscopy 
by Dr. Emma Shelton to verify their cytologi- 
cal composition. The kinetics of labelling ob- 
tained from in vivo incorporation studies 
with C 14 -leucine during incorporation times 
between 1.33 and 11 minutes suggested that 
protein synthesized on the free polyribosomes 
was rapidly transferred in vivo to the soluble 
fraction of the cell, while protein synthesized 
by the microsomes and mitochondria remained 
localized within these elements. In the RPC- 
20 tumor, the free polyribosome fraction and 
the combined microsome-mitochondria frac- 
tion accounted for approximately equal pro- 
portions of the total cytoplasmic protein syn- 
thesis in vivo. 

These results indicated the existence of two 
biosynthetic compartments distinguishable in 
terms of the immediate distribution of their 
protein products. Accordingly, Drs. M. Wolf 
and L. Kedes have undertaken to determine 
whether a corresponding phenomenon could be 
observed during in vitro protein synthesis by 
the isolated fractions, and whether direct ex- 
perimental evidence could be obtained that 
the two compartments are concerned with the 
synthesis of a qualitatively different spectra 
of proteins. Tumors were pulse-labelled in vivo 
and the fractions isolated from them were in- 
cubated in vitro in the presence of soluble frac- 
ton, energy, and a complete C 12 -amino acid 
mixture. Under these conditions, 30-40% of 
the radioactive (nascent) protein of the free 
polyribosomes was transferred to the soluble 
fraction, the release phenomenon being time 
and energy dependent. In contrast, only 10% 
of the labelled protein of the membrane frac- 
tion (microsome plus mitochondria) appeared 
in the soluble fraction, and this transfer ap- 
peared to be non-enzymatic since it occurred 
equally well in the cold and the absence of 
energy. Control experiments have established 



that both fractions do, in fact, incorporate 
amino acid into protein under the in vitro 
conditions employed for the study of nascent 
protein release. The results are thus entirely in 
accord with those obtained in the in vivo ex- 
periments. 

The free polyribosome fraction of the plas- 
ma cell tumor has been studied in some detail 
by electron microscopy by Dr. Emma Shelton. 
The size distribution is generally larger than 
that reported for the polyribosomes of other 
types of tissue, with many polyribosomes com- 
posed of 15-20 monomeric ribosmal units and 
with no evidence that the polyribosomes con- 
tain any organized membrane structure. Sim- 
ilarly, chemical analysis has failed to reveal 
either phospholipid or neutral lipid. Since 
these polyribosomes are active in completion 
and release of nascent peptide chains, their 
protein synthetic activity may be independent 
of lipid components, in contradistinction to 
results reported with other cell types such as 
yeast and regenerating liver. Deoxycholate, a 
detergent in widespread use for isolation of 
polyribosomes from a variety of tissues, has 
a marked effect upon the free polyribosomes 
of the tumor, removing approximately one 
third of their total protein, without, however, 
removing the nascent protein. At the same time, 
the polyribosomes become more sensitive to 
ribonuclease action and less efficient in carry- 
ing out in vitro amino acid incorporation 

An analysis of the structural proteins of var- 
ious mammalian ribosomes by Dr. L. Kedes fol- 
lowed procedures established elsewhere but 
with plasma cell tumor ribosomes as the ini- 
tial objects of study. The complex acrylamide 
gel electrophoretic pattern of the ribosomal 
proteins (up to 35 discrete bands) was identi- 
cal in the case of 9 different tumors (each se- 
creting a different myeloma protein) growing 
in BALB/c mice. The tumor ribosomal pro- 
tein pattern was, in turn, the same as that 
given by the ribosomal proteins of normal 
BALB/c liver. However, the ribosomal pro- 
teins of mouse liver, rat liver, and rabbit retic- 
ulocytes could all be differentiated from one 
another on the basis of their disc electrophor- 
esis patterns. Comparison of the ribosomal pro- 



NATIONAL CANCER INSTITUTE 



17 



teins from inbred mouse strains has been ini- 
tiated. 

Efforts are being made by Dr. M. Wolf to test 
the biological activity of plasma cell tumor 
RNA upon intact cells in which endogenous 
messenger RNA synthesis had been blocked 
by treatment with actinomycin. Although ini- 
tial experiments seemed to indicate some ac- 
tivity (as judged by restoration of protein syn- 
thesis in the treated cells), reproducible results 
could not be obtained. The major problem ap- 
pears to be in finding suitable non-toxic 
substances which, upon complexing with the 
added RNA, will both protect it from enzy- 
matic degradation and facilitate its entrance 
into the target cells. 

Investigation by Mr. N. E. Roberts of the 
non-secretory subline of the RPC-20 plasma 
tumor has continued. It now appears (from 
immunological analysis of tissue fluids that 
were fortunately frozen during the time that 
conversion occurred) that the loss of the ca- 
pacity to produce specific secretory protein 
took place quite abruptly, possibly over a sin- 
gle transplant generation. Attempts to repro- 
duce the phenomenon by continued rapid 
transplantation of the producer line have been 
unsuccessful. Induction of the non-secretory 
subline by a viral infection of the cells has 
not been excluded. 

Dr. M. Rechcigl, Jr. has continued his in- 
vestigation of the physiological role of cata- 
lase and related enzymes and the mechanisms 
of their regulation in normal and neoplastic 
tissues. 

In collaboration with Drs. H. A. Hoffman 
and W. E. Heston, studies on the biochemical 
genetics of catalase have confirmed earlier 
findings that the level of liver catalase activ- 
ity in the mouse is controlled by a single pair 
of genes with low dominant to high. Ce has 
been suggested for the gene symbol. Present 
data indicate that the gene does not act di- 
rectly in the synthesis of the enzyme. The 
presence of the gene reduces catalase actitivy 
to one-half the normal amount rather than 
eliminating it completely and low activity is 
dominant to high in contrast to genetic con- 
trol over direct synthesis of enzymes. Further- 
more, the effect is apparently limited to the 



liver, implying that the gene must be provid- 
ing a regulator of the amount of liver catalase 
activity. This is in sharp contrast with acatala- 
semia in the Japanese, where the gene is ap- 
parently involved directly in the synthesis of 
catalase, for homozygous recessive individuals 
show hardly any catalase in all examined tis- 
sues. On the basis of preliminary data ob- 
tained by determining the rates and the kinet- 
ics of catalase synthesis and destruction in vivo, 
the "regulator gene" action causing the ob- 
served differences in the liver catalase activity 
in the investigated mouse strains is a result of 
the decreased rate of synthesis of the enzyme 
rather than the increased rate of its break- 
down. 

Experiments have been initiated to develop 
a histochemical technique which would permit 
a quantitative or semi-quantitative assessment 
of the relative amount of catalase present in 
individual cells. The general approach was to 
immobilize isolated cells in a thin agar sheet, 
lyse the cells in order to release the contents 
around the cells, and then specifically identify 
regions of high catalase by the ability of the 
enzyme to destroy peroxide, which serves as 
an oxidizing agent in the iodine reaction with 
starch. An assay capable of identifying cata- 
lase-containing cells as visible plaques has been 
developed but no quantitative information has 
yet been obtained. 

Preliminary studies using disc electrophor- 
esis indicate that a variety of tissues may 
contain several isozymes of catalase. The pos- 
sibility of having broken down the catalase 
protein into subunits cannot be overlooked 
though it seems rather unlikely. When the liver 
homogenate supernatants were heated with 
3-amino-l,2,4-triazole, a drug known to destroy 
catalase, the electrophoretic pattern showed 
that one of the "catalases" was inactivated 
while the second catalase was still active. 

Nucleic Acids Section 

The objectves of Dr. W. Schneider's research 
are to isolate and identify the deoxyribose- 
containing compounds present in normal and 
cancer tissues, to determine the metabolic se- 
quences in which they are involved, and to 
study their relationship to DNA synthesis. 



18 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Previous studies on normal liver and the Novi- 
koff hepatoma showed that the levels of dCDP- 
choline and dCDP-ethanolamine were 6 to 7 
times as great in the tumor as in the liver. 
Studies on liver after partial hepatectomy 
now have shown that these compounds also 
increase in amount as early as 12 hours after 
the operation. A single determination on a 
pooled sample of hepatoma 5123, a minimal 
deviation tumor, showed that these compounds 
were also increased several-fold in this tissue. 
The fact that the concentration of these com- 
pounds increases in the liver shortly after par- 
tial hepatectomy, and is high in the minimal 
deviation hepatoma 5123 as well as in the 
highly malignant Novikoff hepatoma, provide 
further evidence for the importance of these 
compounds in growth, although an explana- 
tion for their increased concentration in grow- 
ing tissues remains obscure. The enxymatic 
synthesis of labeled phosphoryl choline, phos- 
choline, dCDP-ethanolamine, and OCDP-ethano- 
lamine has been completed and the results 
published. 

Previous work demonstrated that the phos- 
phoryl choline-cytidyl transferase of rat liver 
was activated by degraded phospholipids. This 
suggested to Drs. W. G. Fiscus and W. Schnei- 
der that a positive feedback mechanism was 
operative in lecithin biosynthesis and led to a 
study of the transferases. Experiments showed 
that a change in size of the enzyme occurred 
in the presence of activating phospholipid and 
indicated that an association of subunits was 
involved in the activation process. In addition, 
the activity of the transferase, in the pres- 
ence of increasing concentrations of CTP, 
showed a sigmoidal curve in the absence of 
activator. In the presence of activator the ac- 
tivity did not show this "lag" phase at low 
concentrations of CPT. Other experiments 
have shown that an inhibitor of the transfer- 
ase is present in rat liver particulate fractions 
and can be released into solution by boiling 
the particulate suspension. This inhibitory 
factor appears to be bucleotide in nature. If 
labeled CDP-choline is incubated with this 
fraction and a liver homogenate, the cytosine 
moiety of the CDP-choline is incorporated into 



a material which is also inhibitory to the trans- 
ferase and which may be a polynucleotide. 

These results suggest that CDP-choline is 
involved not only in phospholipid formation 
but may also be involved in the formation of 
polynucleotides. Of interest from this stand- 
point is the observation that dCDP-choline, 
but not CTP, CDP, or CMP, dilute the incorpo- 
ration of CDP-choline into the polynucleotide. 

Furthermore, the present studies indicate 
that the transferase belongs to the group of 
allosteric enzymes since its activity depended 
upon the presence of phospholipid activator, 
its conformation changed during activation 
possibly due to an association of subunits, its 
substrate concentration curve was sigmoidal 
in the absence of activator, and it was inhib- 
ited by a naturally occurring material which 
appears to be a polynucleotide. 

The work on DNA in isolated liver mito- 
chondria, begun last year by Dr. W. Schneider, 
was continued and some of the results have 
been published. A method was developed for 
isolating DNA from both nuclei and mitochon- 
dria in highly purified form. Tests of these 
DNA samples by buoyant density determina- 
tions in CsCl and by melting point determina- 
tions confirmed the results reported last year 
by direct base analyses and show that the 
guanine-cytosine content of the mitochondrial 
DNA was 2 to 3 percent lower than that of 
the nuclear DNA. In addition, these studies 
also showed that the mitochondrial DNA was 
of large size with a molecular weight of 8 to 9 
million. The mitochondrial DNA also appeared 
to be more homogeneous than the nuclear DNA, 
as judged by the sharpness of the bands ob- 
tained in the CsCl centrifugations and the 
temperature range required to melt the nu- 
cleic acid samples. Another difference between 
the nuclear and mitochondrial DNA was in 
the rate at which labeled thymidine and de- 
oxycytidine were incorporated in vivo. Both 
of the precursors were found to be incorpo- 
rated to about 10 times as great an extent 
into liver mitochondrial DNA as into nuclear 
DNA in adult rats. More recent experiments 
have shown that although the incorporation 
into nuclear DNA was increased enormously 
24 hours after partial hepatectomy, the incor- 



NATIONAL CANCER INSTITUTE 



19 



poration into mitochondrial DNA was unaf- 
fected. A study of the retention of label in the 
mitochondrial DNA showed that the specific 
activity of the mitochondrial DNA fell off rap- 
idly, with a half life of about 7 days. 

Preliminary experiments have also been car- 
ried out on the livers of rats fed the potent 
carcinogen, 3'-methyl-N,N-dimethylaminoazo- 
benzene (3'-MeDAB) or the non-carcinogen, 
2-methyl-N,N-dimethylaminoazobenzene (2- 
MeDAB) for 14 and 28 days. The expected in- 
crease in liver mitochondria in the rats fed the 
2-MeDAB and the expected decrease in liver 
mitochondria from rats fed the 3'-MeDAB 
were observed but the rate of incorporation of 
thymidine into the mitochondrial DNA did not 
seem to differ in these rats and in controls. The 
most significant finding of these experiments 
was a 2-fold increase in DNA in the 3'-Me- 
DAB liver mitochondria. It seems likely that 
the study of much shorter periods of feeding 
these dyes will be necessary to determine 
whether the mitochondrial DNA is affected 
by either dye. 

These studies on mitochondrial DNA pro- 
vide a new point of departure for experiments 
in cancer research. Since experiments per- 
formed here, as well as elsewhere, have shown 
that tumors contain fewer mitochondria and 
have decreased levels of mitochondrial en- 
zymes and that these changes in mitochondria 
occur in the liver in the earliest stages of chem- 
ical carcinogenesis, it seems logical to ask 
whether the point of attack of the carcinogen 
is at the mitochondrial DNA. Experiments on 
this point are in progress. 

During the past few months Dr. S. Lerner 
and Dr. W. Schneider have begun to examine by 
hybridization techniques the degree of com- 
plementarity that may exist between DNA and 
RNA isolated frcm rat liver mitochondria. 
Heretofore, efforts have been directed mainly 
toward the isolation and clean separation of 
DNA and RNA. If, however, mitochondrial 
RNA is found to hybridize with the DNA, 
this would suggest a transfer of information 
from the DNA, perhaps analogous to the RNA- 
mediated expression of information in protein 
synthesis, and would provide impetus for the 
investigation of possible autonomy for mito- 



chondrial biosynthesis which endogenous DNA 
might afford. 

Partial purification of TMP kinase free of 
TMP phosphatase activity was achieved by 
Dr. R. K. Kielley with successive steps involv- 
ing pH 4.8 precipitation, (NH 4 ) 2 S0 4 fraction- 
ation of soluble protein, and treatment with 
DEAE-Sephadex. 

TMP-phosphatase activity was totally ab- 
sent in the final product. The specific activity 
of the purified kinase is the highest reported 
from mammalian tissue, but the purified en- 
zyme is still contaminated with some ATPase. 
TdR kinase, diphosphokinase and myokinase 
activity, as determined by spectrophotometry, 
coupled enzyme assays. Other purification pro- 
cedures with Sephadex gel filtration and CM- 
Sephadex have been investigated and shown 
to be feasible. Removal of the protective sub- 
strate (2xlO~°mTMP) from the enzyme 
by passage through Sephadex G-25 led to in- 
activation, the degree depending on the time 
required for passage, suggesting a dissociable 
complex of the enzyme and protector. The 
kinetics of the time course of inactivation in- 
dicated a latent period lasting several minutes 
during which time an intermediate may be 
formed. It has been shown that TMP kinase, 
an enzyme which plays a key role in DNA 
synthesis, has a sulfhydryl requirement for 
stability, a requirement not detected in crude 
extracts of the enzyme. Pre-incubation expe- 
riments indicated that glutathione conferred 
increased stability to enzyme already pro- 
tected with TMP. 

It is noteworthy that stability in the presence 
of ATP occurred only when GSH was present. 
A tentative explanation might be that the ac- 
tual stabilizing nucleotide bound to the enzyme 
is the reaction product itself, TDP, rather than 
the substrate, TMP, which appeal's to be less 
effective. The GSH effect may serve to increase 
the binding strength of the enzyme for TDP 
to such an extent that the "bound TDP" is 
unavailable for further phosphorylation even 
though diphosphokinase and ATP are present 
to convert TDP to TTP. 

The nature and extent of the biochemical re- 
actions by which nucleic acid precursors are 
metabolized in cultured nonmalignant and neo- 



20 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



plastic cells are being studied by Dr. J. Rother- 
ham, particularly with respect to their possible 
significance in nucleic acid synthesis and neo- 
plastic transformation. 

It has been demonstrated that a strain of 
C3H mouse embryo cells (Tissue Culture Sec- 
tion, Laboratory of Biology) grown in chemi- 
cally-defined medium has a nutritional require- 
ment for thymidine. The thymidine can be 
replaced by doexycytidine or 5-methyldeoxycy- 
tidine but not by uridine, deoxyuridine or cyti- 
dine. Thus, it appears that the deoxynucleo- 
sides which were utilized have a significant 
metabolic function for this cell strain and that 
biochemical reactions by which the latter 
three nucleosides can be converted to the for- 
mer were not taking place. An explanation for 
these findings is being sought frcm tracer ex- 
periments in which the cells are exposed to 
C 14 -labeled nucleosides in the medium. From 
isolation of the nucleoside products found in 
the medium and in the DNA or RNA of the 
cells and from a determination of the approxi- 
mate extent of conversion of precursors to 
product it can be concluded that there is a 
rapid deamination of decxycytidine and/or 
phosphorylation followed by deamination and 
dephosphorylation — and that the deoxycyti- 
dine can also be phosphorylated and utilized 
for synthesis of DNA-deoxycytidine and/or 
thymidine. 

As part of a continuing study of the func- 
tion and relationships of the structure and se- 
quence of nucleic acids, the nucleotide distri- 
bution in nucleic acids, the development of 
new fractionation procedures, and the prepa- 
ration and characterization of nucleotide se- 
quences are under investigation by Drs. G. W. 
Rushizky and H. A. Sober. Various protein 
fractionation techniques are utilized for the 
isolation, from sources such as micro-organ- 
isms, of nucleases (both of RNases and DN- 
ses) with hydrolytic activities directed 
toward specific nucleotide linkages. Since DN- 
ases, in particular, are quite unstable, new 
techniques have to be developed. A group of 
DNases have been characterized and found to 
consist of a mixture of at least 2 endonucle- 
ases and 1 phosphodiesterase. The latter en- 
zyme hydrolyzes not only DNA but also RNA. 



Separation of the 2 endonucleases by standard 
protein fractionation techniques has not met 
with success since loss of the major portion 
of the enzymatic activity resulted. These DN- 
ases are obtained from the mold Aspergillus 
oryzae, which is also the source of the Very 
useful enzymes, RNases T 1 and T 2 . Other pre- 
viously described RNases have been prepared 
from B. subtilis, B. cereus, and various molds, 
and the possible presence of new DNases has 
been investigated. A RNase-free phosphatase 
from E. coli C90 has been prepared. 

There is always a need for new methods for 
the separation, and characterization of oligo- 
mers of mixed base ratios ranging in size from 
10-100. To date, a supply of such large olig- 
omers is one of the limiting factors in design- 
ing such procedures. As a source of large oli- 
gonucleotides, MS2 phage RNA (chain length 
of 3,000 nucleotides) was purified in good 
yield. Octa- and decanucleotides from complete 
RNase T, digests of MS2 RNA were fraction- 
ated according to their (Ap + Cp): Up content 
by mapping or column chromatography at pH 
2.7 in 7 M urea. Penta-, hexa-, and heptamer 
mixtures of identical Up (and Gp) composition 
were separated according to Ap:Cp ratios by 
the same procedures but at pH 4.3. Such oli- 
gomers have been examined for hyperchromy 
and optical rotatory dispersion. Even larger 
oligomers have been obtained by digestion of 
of MS2 RNA after it had been absorbed on 
DEAE-cellulcse, yielding groups of oligomers 
of chain length 14 to 400 with 51 to 84% re- 
covery of the starting material. Oligomers de- 
rived in this manner by Micrococcal nuclease 
digestion had base ratios very similar to those 
of the original MS2 RNA. On the other hand, 
oligonmers with Cp:Gp ratios of 2:1 were ob- 
tained when B. subtilis RNase was used as the 
enzyme (the ratio is approximately 1:1 in MS2 
RNA). Preliminary results indicate that this 
procedure also is applicable to the isolation of 
large oligomers from DNA. 

These procedures have been used to prepare 
pure oligonucleotides of known chain length 
and nucleotide sequence and has permitted a 
study of their chemical, physical and biological 
properties. Oligomers so prepared have been 
used in studies of nuclease specificity, neigh- 



NATIONAL CANCER INSTITUTE 



21 



bor-neighbor interactions in single oligonucle- 
otide strands, and as test materials for com- 
putational analysis of nucleic acid spectra. 
Deoxynucleo tides, greater than or equal to 7 
in chain length, have produced reactivation 
of the immune response in thymectomized, 
X-ray irradiated adult mice in exploratory ex- 
periments with Dr. M. Feldman (Weizmann 
Institute of Science). Attempts to duplicate 
the result in in vitro spleen cultures have so 
far been unsuccessful. 

Protein Chemistry Section 

The polylysine-polynucleotide system was 
chosen by Dr. H. A. Sober as a simple model 
of nucleoprotein interaction. This interaction 
is known to affect the biological function of 
the nucleic acid. A study of the nature of the 
binding forces, stoichiometry, and specificity 
of this interaction should shed some light on 
the mechanism by which a basic protein "rec- 
ognizes" a portion of the nucleic acid structure 
and affects its properties. 

Polylysines react on a molar basis with RNA 
to form insoluble complexes at low salt concen- 
trations and neutral pH. Soluble complexes 
are formed, however, at low polylysine-to-RNA 
ratios. The soluble complexes are susceptible to 
hydrolytic degradation by specific and non- 
specific ribonucleases with release of soluble 
nucleotides. Digestion continues until the for- 
mation of a precipitate with a nucleotide-to- 
lysine ratio of one. After dissociation of the pre- 
cipitated complex and separation of its com- 
ponents, the "protected" nucleotide chain is 
again susceptible to cleavage by the original 
enzyme. The protected nucleotide sequence is 
shorter than that of the original RNA. Thus, 
treatment with polylysine, n = 100, results in 
a protected RNA sequence of n = 99; the use 
of oligolysine (n = 13) results in a RNA seg- 
ment of n = 16. Oligolysines smaller than the 
heptamer do not form enzyme-resistant com- 
plexes. 

Present evidence indicates that the nucleo- 
tide composition of the protected sequence 
may be markedly different from that of the 
parent molecule and that overlapping nucleo- 
tide sequences may be obtained by this pro- 
cedure. While no base specificity has been found 



with heated or "denatured" RNA, with "na- 
tive" or unheated RNA, the protected RNA 
segments contain an altered proportion of 
gyanylic (G) and cytidylic (C) acid residues 
as much as 76% (G + C). 

Measurements by Dr. S. Latt have shown 
that the binding of oligolysine to MS2 RNA, 
Poly (I + C), and Poly (A + U) was reversible 
as long as the polynucleotide-oligolysine com- 
plex remained soluble. Preliminary data with 
the single homopolymers show precipitation 
even at very low free oligolysine concentra- 
tions. Binding of oligolysine to polyribonu- 
cleotide decreases markedly with increasing 
ionic stength, and is virtually nonexistent in 
1.5 M NaCl, indicating that the major if not 
sole binding force is electrostatic. Binding 
strength increases markedly with oligolysine 
chain length. An equation for reversible bind- 
ing of a linear oligomer to a much larger lin- 
ear polymer was derived and experimental 
binding curves obtained were of the form pre- 
dicted. 

Another approach to nucleoprotein interac- 
tion is a new project by Dr. 0. W. McBride 
wherein the role of nucleic acid-bound proteins 
in the regulation of the transcription and 
translation of genetic information will be ex- 
amined. Studies have been initiated recently 
to determine the most suitable procedures for 
isolating soluble chromosomal nucleoproteins 
from rat liver in a physical condition closely re- 
sembling the native state of these nuclear 
components. A variety of modifications of two 
basically different procedures are being exam- 
ined. These methods involve either the direct 
isolation and purification of the chromosomal 
apparatus from aqueous solutions or isolation 
and extensive purification of whole nuclei 
prior to isolation of soluble nucleoproteins. 
The criteria which have been established for a 
satisfactory procedure are the isolation of a 
soluble nucleoprotein preparation without deg- 
radation or with minimal, controlled, and re- 
producible degradative methods and the ab- 
sence of dissociation, association or exchange 
of chromosomal components with either cyto- 
plasmic or nuclear non-chromosomal molecules. 
The possible exchange between soluble his- 
tones or non-histone proteins as well as be- 



22 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tween soluble ribonucleic acids and nucleopro- 
teins will be examined under the specific ionic 
conditions of the isolation procedure with iso- 
topically-labeled material. Rat liver nucleopro- 
teins will be fractionated by a combination of 
several independent methods utilizing differ- 
ences in mass, buoyant density, electrostatic 
charge, and surface characteristics of these 
particles. The fractions will be examined for 
differences in ribonucleic acids, histones, non- 
histone proteins, and lipids associated with 
these DNA segments. Covalent and non-cova- 
lent associations between these various com- 
ponents will be investigated to obtain some in- 
sign concerning the particular component or 
components which may be responsible for a 
specific association with the nucleic acid. 

Attempts to purify the Moloney virus, which 
is known to produce a generalized lymphocytic 
neoplasm in mice and rats were made by Dr. 
C. W. Lees and Dr. H. A. Sober with the col- 
laboration of Dr. J. B. Moloney (Laboratory 
of Viral Biology). Fractionation of viral ac- 
tivity was attempted by ion-exchange chroma- 
tography, Sephadex chromatography, and the 
aqueous phase liquid-liquid partition system of 
Albertsson. While apparent purification of sev- 
eral-fold was obtained, reproducibility of the 
assays was inadequate to confirm these results 
or to permit the desired estimate of the re- 
covery of activity. These exploratory studies 
were designed to indicate the range within 
which fractionation procedures must remain 
in order to obtain good yields of active virus. 
Because of the large number of fractions which 
develop during fractionation, a "rapid" and 
reasonably quantitative assay procedure must 
be available. The 4-week bioassay used in these 
experiments does not satisfy these require- 
ments. 

Dr. R. W. Hartley, Jr. with the collaboration 
of Dr. C. W. Lees has been ivolved in the puri- 
fication and characterization of an extra cellu- 
lar ribonuclease of B. subtilis by physical and 
chemical techniques and the development of 
methods of culture and genetic analysis so that 
the organism and its enzyme product may be 
applied to a long range study of enzyme struc- 
ture, function, and synthesis. 

A strain of B. subtilis has been derived from 



strain H that will grow in a synthetic medium 
and produce ribonuclease on a continuous 
basis. This was achieved by an evolutionary 
process in a continuous fermenter. Experience 
with a small (100-200 ml per stage) two stage 
continuous fermenter has established the feas- 
ibility of producing crude enzyme in a contin- 
uous and semi-automatic fashion in such an 
apparatus. Production at a rate of at least 100 
to 200 mg of crude enzyme per day is envis- 
aged with a 14 liter per stage set-up cur- 
rently available. 

Amino acid analysis of the purified enzyme 
has confirmed the absence of sulfur amino 
acids except for 0.2 moles per mole of methi- 
onine which is probably part of a contaminant. 
All of the weight and nitrogen of the enzyme 
was accounted for by the amino acid recov- 
ery. Dry wevht, nitrogen and U.V. absorp- 
tion determinations gave a nitrogen content 
of 17.7% and an E 1%/1 cm at 280 m/iof 20.9 
The high extinction is in line with the high 
tryptophan content. Quantitative N-terminal 
analysis by dinitrophenylation in two experi- 
ments showed 0.7 and 0.9 mole of N-terminal 
alanine per mole of enzyme and no other N- 
terminal residues. Alanine alone was also 
found by the DNS ("dansylation" with di- 
methylnapthalene-sulfonyl chloride) method. 
The molecular weight of 10,700 was deter- 
mined by ultracentrifugal analysis. 

The enzyme is absorbed in considerable quan- 
tity by glass and other surfaces from water 
and many buffer systems. This is largely pre- 
vented by di- and tri-valent cations, especially 
iron, by amines such as choline and cetyltri- 
methylammonium bromide, and, fortunately, 
by 0.1 M ammonium bicarbonate which will 
quantitatively release enzyme adsorbed from 
0.1 M Tris hydrochloride solution but not en- 
zyme adsorbed from water or washed with 
water. 

The dry enzyme is insensitive to heat 
(100°). In solution (0.1 M NH 4 HC0 3 ) how- 
ever, 90% of the activity is lost within an hour 
at 90° with significant loss of activity even 
at 60°. The protein remains in solution, but 
moves as a discrete band on disc electrophore- 
sis at pH 4.5 behind that of the active enzyme, 
suggesting that the loss of activity is due to 



NATIONAL CANCER INSTITUTE 



23 



hydrolysis of amide groups. The inactive en- 
zyme still reacts with a specific B. mbtilis in- 
tracellular ribonuclease inhibitor, preventing 
inhibition of added active enzymes. Activity 
remains constant in 1 2V HC1 at 0° up to 24 
hours but is largely lost in one hour at room 
temperature. In 1 N NaOH at 0°, about 30% 
of the activity remains at 1 hour and very 
little remains at 4 hours. 

Several ribonuclease-negative mutants have 
been isolated as well as a larger number of 
protease- and amylase-negative mutants. 

Dr. E. A. Peterson and Dr. W. H. Evans have 
undertaken to develop methods for the frac- 
tionation of normal and leukemic blood and 
bone marrow leukocytes as a basis for the 
study of the fundamental biochemical changes 
that underlie the maturation of normal leu- 
kocytes and the factors that lead to the arrest 
of maturation in leukemia. 

Several stages of maturation in the ery- 
throid and myeloid series of guinea pig bone 
marrow cells have been partially resolved by 
sedimentation in a dilute density gradient at 
unit gravity in a simple apparatus. Besides the 
clear separation of erythrocytes from the my- 
eloid cells, a useful separation of blasts, neu- 
trophil myelocytes, and mature neutrophils 
was achieved, although the overlap of peaks 
left something to be desired. Lymphocytes 
emerged with the erythroid cells but were well 
separated from all other types except blasts. 
Peak enrichments of given cell types ranged 
from 3- to 15-fold. The time required for such 
resolution varied from 5 to 20 hours at 4°. 
However, the myeloid cells were 92 percent 
viable after 20 hours, as determined by the 
trypan blue method. Erythrocyte precursors 
were only 41 percent viable after this period 
and 67 percent viable after 5 hours. Although 
migration was almost linear with time, useful 
resolution did not increase in proportion to the 
time allowed for sedimentation under the same 
conditions, indicating an overlapping hetero- 
geneity of the cell classifications with respect 
to sedimentation rate. The medium employed 
was a Krebs-Ringer phosphate solution from 
which calcium and magnesium were omitted 
and to which 0.01 percent polyacrylate was 
added. These changes were necessary to pre- 



vent reaggregation of the cells after they were 
dispersed. The presence of polyacrylate did not 
affect the viability count. 

Studies of the behavior of guinea pig bone 
marrow cells on passage through slowly rotat- 
ing horizontal columns of open cell polyure- 
thane foam have been continued. Enzyme 
treatment of the cells in order to prevent non- 
specific retardation of a considerable portion 
of the myeloid cells in columns of foam having 
100 pores per inch was mentioned in last year's 
report. A similar effect has now been achieved 
by dispersing the cells in a medium contain- 
ing polyacrylate and treating the foam with 
the same substance. Whether it is bound to 
the cell or merely cleans it (of, e.g., nucleopro- 
tein) is now known at present, but polyacry- 
late is bound to the foam. Similarly, these 
foams have been shown to bind polyethylene- 
imine and tetraethylene-pentamine. Moreover, 
foams treated with these polyamines strongly 
adsorb guinea pig bone marrow cells, includ- 
ing the red cells which pass unadsorbed 
through untreated foams under the same con- 
ditions. Thus, the foams can be converted 
readily into ion exchanges. Presumably, the 
polyacrylic acid chain is bound when one or 
more of its many carboxyl groups engages in 
an acid-exchange reaction with the polyester 
moiety of the particular polyurethane foams 
used in this work. The polyamine chains are 
similarly bound when one or more of the 
amino groups cleaves the polyester to form 
an amide bond. Theoretically, many substances 
can be attached to the foam in this way, pro- 
viding the possibility of readily tailoring the 
nature of the surface for a wide variety of pur- 
poses. Among others successfully bound were 
proteins such as histones, protamine, gamma- 
globulin, and albumin. 

The ability to bind gamma-globulin to the 
foam columns suggested the feasibility of uti- 
lizing immunological affinity for the separa- 
tion of cells, provided the bound gamma- 
globulin retained its activity. In collaboration 
with Dr. Michael Mage (NIDR) a study of such 
a possibility was undertaken, using the circu- 
lating red cells of the guinea pig as a model 
antigen. These experiments were carried out 
at room temperature (25°) in order to pro- 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mote a reasonably rapid immunological reac- 
tion. It was found that the circulating red cells 
were bound to untreated foam, presumably 
through the sialic acid on their surfaces, but 
they were readily dislodged by streams of li- 
quid, by very low-power sonication, or by 
squeezing the foam. Treatment of the foam 
with normal rabbit gamma-globulin prevented 
this binding of red cells, apparently by cover- 
ing the reactive sites. In contrast, foams 
treated with specific rabbit gamma-globulin 
(anti-guinea pig RBC) at the same protein 
concentration adsorbed the red cells entering 
the column and became bright red. Again, the 
bound red cells could be completely removed 
from the foam by the low shear applied by 
the moving liquid when the foam was squeezed. 
It is not known, as yet, whether the release of 
the cells involved dissociation of the antigen- 
antibody complex or cleavage of some other 
linkage, but the cleaned foam could be used re- 
peately to bind red cells. Similarly, the foam 
protected with normal gamma-globulin retained 
its protection through repeated uses. The spe- 
cificity of this interaction has not as yet been 
completely established, but sheep cells were 
not bound by the anti-guinea pig RBC foam. 
On the other hand, an anomalous low-inten- 
sity interaction between rabbit RBC's and nor- 
mal rabbit gamma-globulin and a strong in- 
teraction between rabbit RBC's and normal 
bovine gamma-globulin have been observed. 

The discovery that the polyester-based poly- 
urethane open-cell foams can readily be given 
a wide range of ion-exchange and immunolog- 
ical properties opens the way for the develop- 
ment of procedures for cell separation based 
on such principles. Studies of the nature of 
cell surfaces should also be aided, and appli- 
cations in basic immunological investigations 
are foreseen, particularly in view of the po- 
tential ability of this type of system to detect 
and measure monovalent as well as multivalent 
antibodies. 

Studies on the basis of heterogeneity of an- 
tibody formed to single, well-defined antigens 
have been performed by Dr. S. Schlossman 
(Beth Israel Hospital, Boston, Mass.) and 
Dr. H. A. Sober. A homologous series of com- 
pounds was used because the chemical defini- 



tion both in position of the hapten and in pep- 
tide chain length provide obvious advantages 
over less well-defined materials in studies deal- 
ing with the chemical basis of the immune 
response. 

Immunogenicity was observed in Hartley 
and strain 2 guinea pigs with a, N-DNP-hepta-, 
octa, and nona-L-lysine, whereas smaller a, 
N-DNP-L-lysines were not immunogenic. The 
L configuration and the presence of a hapten 
were also required for immunogenicity in this 
system. The same antigen, e.g., aiV-DNP-octa- 
L-lysine, induced the formation of both de- 
layed and immediate sensitivity. Using chem- 
ically defined «-DNP(Lys) n BuAm peptides, 
related peptides, and hapten-substituted pro- 
teins, only the immediate skin response (Ar- 
thus) could be elicited with hapten-substituted 
tetra-, penta-, or haxmers, whereas both imme- 
diate and delayed skin responses could be pro- 
voked by the octamer or nonamer. The hapten 
is an integral part of the determinant for both 
immediate and delayed skin reactivity, since 
poly-L-lysine was unable to elicit either reac- 
tion in sensitized animals. Immediate-type 
cross reactions occurred whenever the sensi- 
tizing and test antigen shared a common hap- 
tenic determinant. In contrast to this, in this 
system, delayed-type cross reactions occurred 
only when the test antigen and the sensitizing 
antigen contained both a large oligo-L-lysine 
carrier as well as the same haptenic determi- 
nant. 

These observations imply that the media- 
tion of the delayed response requires a larger 
determinant than is necessary to elicit the im- 
mediate response. High affinity antibody as 
the mediator of the delayed response is not con- 
sidered a satisfactory explanation of the ob- 
served phenomena. However, the fact that the 
ability to elicit the delayed response parallels 
the immunogenic capacity of these pepties 
suggests that the delayed response may re- 
quire the continued biosynthesis of antibody 
and may be analogous to a local in vivo sec- 
ondary response. 

The properties of poly-a-amino acids, model 
substances closely analogous to proteins, are 
being studied by Dr. H. A. Sober and M. C. 
Otey with the collaboration of Drs. A. Berger, 



NATIONAL CANCER INSTITUTE 



25 



A. Yaron and E. Katchalski (Weizmann In- 
stitute of Science, Rehovoth, Israel). Oligoly- 
syl peptides, in particular, are useful model 
compounds for the investigation of the inter- 
action and functions of the basic proteins. 
These histone analogs, now available in chain 
lengths ranging from 2 to 25 residues long 
have been used in a number of studies de- 
scribed above, namely: interaction with nu- 
cleic acids and polynucleotides to form nu- 
clease-resistant complexes, where the length 
of RNA segment is directly related to the 
length of oligolysine used; after mono- and di- 
hapten addition, provide a chemically-defmed 
series ranging from no- to full antigenicity 
which has been used to define the antigenic 
determinants of delayed and immediate skin 
sensitivity; optical rotatory dispersion studies 
with Dr. G. Fasman (Brandeis University), 
examining the effect of chain length on pro- 
tein conformation; and the variation in chemi- 
cal properties, such as the apparent pK of the 
e-amino group, the color yield with ninhydrin, 
and adsorption to polyanionic substances as 
the oligopeptide is extended. 

It has long been apparent that metals pres- 
ent in blood and tissues function in association 
with proteins, and whenever it has been pos- 
sible to examine this association, areas of ma- 
jor biochemical importance have been uncov- 
ered. By collaboration between the Laboratory 
of Biochemistry, NCI, and the Biophysics Lab- 
oratory (Peter Bent Brigham Hospital, Har- 
vard Medical School, Boston, Mass.), a com- 
bination of spectrographic analyses and 
protein fractionation has been developed which 
makes it possible to isolate and study the asso- 
ciation of metals and proteins in much greater 
detail. 

Several new zinc proteins have been recog- 
nized in serum by Dr. S. R. Himmelhoch using 
these procedures. In view of the abnormality 
of serum zinc concentration reported in Laen- 
nec's cirrhosis and in leukemia, the existence 
of these three zinc proteins is of special inter- 
est. In Laennec's cirrhosis at least, preliminary 
studies indicate that serum zinc alteration in 
this disease is a reflection of changes in the 
relative amounts of the firmly bound zinc 
moieties. Studies are in progress to complete 



the isolation of these proteins and discern 
their functional role. 

Earlier studies, using limited methods of 
protein fractionation had shown that a pro- 
tein could be prepared from human or horse 
renal cortex which contained about 30 resi- 
dues percent cysteine, and 5% cadmium and 
2% zinc by weight. By application of the 
techniques developed from the above-men- 
tioned collaboration, Drs. S. R. Himmelhoch 
and N. H. R. Kagi and B. L. Valley (Harvard 
Medical School) have been able to show that 
this protein, metallothionein, is but one mem- 
ber of a family of at least four similar pro- 
teins of related amino acid composition, that 
these proteins occur in the supernatant frac- 
tion of renal cortical cells, and that they con- 
stitute 3% of the total protein in this source. 
Methods for preparation of 1 gram quantities 
of each of these components from both equine 
and human sources with quantitative recover- 
ies have been developed. Two additional cad- 
mium binding moieties, one of larger and the 
other of smaller molecular weight than the 
"metallothioneins" have been identified. To- 
gether these proteins account for 98% or more 
of renal cadmium content. Other metals of 
great interest in humanpathology, including 
lead and mercury, have been found in asso- 
ciation with seme of these moieties. The quan- 
titative amino acid composition of these pro- 
teins has been determined and demonstrates 
distinct differences in detail, but a common 
high cysteine content. 

The metallothioneins, a family of peculiar 
cadmium- and zinc-containing proteins, repre- 
sent an important portion of horse and human 
renal cortical cellular protein. Their unique 
structure, with every third amino acid pos- 
sessing a reactive sulfhydryl group, suggests 
a role in renal transport or detoxification pro- 
cesses. Their unusual metal content and small 
molecular weight provide a unique opportunity 
to study metal protein interaction in a defini- 
tive manner. Its role in human disease is under 
study. 

Fractionation of human serum on DEAE- 
cellulose had originally suggested that magne- 
sium might be associated with proteins of 
gamma-globulin mobility. However, further 



26 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



studies by Dr. N. A. Cummings point to a loose 
binding between the metal and proteins, i.e., 
the existence of a magnesium-protein complex 
rather than a metalloprotein per se. By com- 
bining analytic and preparative ultracentri- 
fugation, in collaboration with Dr. E. L. Kuff 
about 70% of the serum magnesium is found 
to be free, with the remaining 30% in a weak 
magnesium-albumin association. The informa- 
tion obtained indicates that magnesium in se- 
rum exists in a locsely-bound protein form, 
capable of being transported and easily re- 
leased by mild changes in salt concentration 
or pH. 

Cryoglobulins isolated by cold precipitation 
from sera have been further purified by gel 
filtration by Dr. N. A. Cummings and have been 
examined by physico-chemical methods in or- 
der to study the phenomenon of cryo-precipi- 
tation. The solubility characteristics of 7S-cry» 
oglobulins are similar to those of normal IgG 
globulins except for temperature effects. Cry- 
oglobulin solubility is a linear function of 
temperature. Although hydrogen-ion titrations 
failed to reveal significant differences in sur- 
face charges at different temperatures, meas- 
urements of viscosity, sedimentation, and sed- 
imentation equilibrium indicated a probable 
unfolding or anisotropic swelling of the mole- 
cule with rising temperature. Further studies 
to elucidate conformational changes will be 
undertaken, including optical rotatory disper- 
sion. 

LABORATORY OF BIOLOGY 

During the year the Laboratory of Biology 
has continued to make significant advances in 
the basic areas of biologic research as related 
to cancer. The program has been expanded 
particularly in the areas of protein chemistry, 
electron microscopy, experimental pathology, 
and cytogenetics. 

Progress is being made in bringing in new 
personnel to strengthen certain promising 
areas of research. We have also lost a few 
members. Dr. Morris K. Barrett retired in July 
and Dr. Margaret K. Barrett resigned in Au- 
gust. Dr. Charles Nicoll resigned in January to 
return to the University of California in Berke- 



ley, where he accepted a position in the De- 
partment of Physiology. 

Dr. William T. Hall, an electron microscop- 
ist, joined the Laboratory as a biologist in Sep- 
tember and now has his electron microscope 
and laboratory set up and has an active re- 
search program underway. Dr. Ettore Appella, 
a protein chemist, got his A.C.S. Fellowship 
renewed and joined the staff in the Carcino- 
genesis Section in September. He has been 
very actively engaged on the amino acid se- 
quences of the myeoloma immunoglobulins 
that Dr. Potter has been studying. Request for 
his appointment next September as a Visiting 
Associate has been submitted. Dr. Ram Par- 
shad, a cyto geneticist, joined the Tissue Cul- 
ture Section in November as an International 
Fellow. He is making significant contribution 
in studying the chromosomes of their cells in 
culture. Three new Research Associates joined 
the Laboratory in July, Dr. Hewes Agnew who 
is working with Dr. Law; Dr. Frederic Mushin- 
ski who is working with Dr. Potter; and Dr. 
Gerald Mackler who is working with Dr. Evans. 

Dr. John T. Mitchell, a developmental biolo- 
gist, is expected to join the staff of the Tissue 
Culture Section in May as a Staff Fellow, and 
appointment of Dr. Raymond Gantt, a bio- 
chemist, also as a Staff Fellow in Tissue Cul- 
ture has been approved for September. Dr. M. 
Ben-David from Hebrew University has been 
appointed to the staff of the Laboratory as a 
Visiting Scientist to report July 1st. He is an 
endocrinologist who is replacing Dr. Nicoll. 
His principal interest is prolactin and we hope 
to investigate the role of this hormone in 
mammary tumorigenesis. Two new Reserach 
Associates, Dr. Ruffner and Dr. Burstein, are 
joining the Laboratory July 1, replacing Dr. 
Granner and Dr. Lyon. This will make a total 
of 72 on the Staff of the Laboratory. 

No attempt is made to mention all findings 
in this summary, but examples are chosen to 
illustrate the scope of the program of the 
Laboratory under the following headings. 

The Thymus in Immunology 

Dr. Law is continuing the characterization 
of the thymic humoral factor that is respon- 
sible for initiating and maintaining lymphoid 



NATIONAL CANCER INSTITUTE 



27 



cell homeostasis and immunologic competence. 
This has been demonstrated in his studies on 
effect of neonatal thymectomy. One new ob- 
servation is that spleen cells from C57BL mice 
produce "runt disease" when injected into 
strain A mice because of the graft versus host 
reaction, but if the spleen cells are from a 
thymectomized C57BL they lack the immuno- 
logic competence to induce the graft versus 
host reaction. Syngenic thymic grafts in thy- 
mectomized C57BL mice restore this compe- 
tence of the spleen cells, but dissociated thy- 
mic grafts do not. The relation of this area 
of research to viral carcinogenesis is summar- 
ized in the next section. 

Tumor Viruses 

The observation that neonatal thymectomy 
can increase oncogenesis by certain viruses has 
been extended by Law, Ting, Agnew, and col- 
laborators. Strain A mice that have never 
been shown to develop tumors of the salivary 
glands when infected with polyoma virus do 
show such neoplasms as well as those of bone, 
mammary, hair follicle, and subcutaneous tis- 
sue when the infection with virus is preceded 
by neonatal thymectomy. Although the Mo- 
loney murine sarcomogenic virus induces tu- 
mors in mice it does not in newborn intact 
rats, but when the rats were neonatally thy- 
mectomized and then inoculated with the virus 
30 to 50 percent of them did develop tumors 
at the site of inoculation. Neonatal thymec- 
tomy did not increase but actually reduced the 
occurrence of mammary tumors in C3H fe- 
male mice with the mammary tumor virus but 
made no difference in the occurrence of MGA 
induced sarcomas in C3H or C57BL mice. 
Furthermore, neonatal thymectomy has made 
no difference in the occurrence of MCA in- 
duced lung tumors in suceptible (C3HfxA)F! 
hybrid mide or in resistant (C3HxB)F 1 hy- 
brids, as shown by their collaborative study 
with Heston. These observations would indi- 
cate that the thymus is responsible for an im- 
munologic mechanism of the homograft type 
that causes target cells to resist oncogenesis 
by certain viruses such as polyoma and MSV, 
but with certain exceptions as the MTV. It is 
suggested that no virus is involved in MCA 



induced sarcomas and lung tumors and thus 
they are not influenced by thymectomy. 

Andervont has continued his observations of 
the activity of the mammary tumor virus 
MTV from strain RIII mice as compared with 
that of MTV from C3H mice when transmit- 
ted normally through the milk. The RIII MTV 
is a relatively weak virus that occasionally 
disappears or becomes inactive in RIII mice. 
When transmitted to agent free C3H mice the 
RIII MTV may also disappear, and when re- 
tained does not increase in activity. The high- 
ly active C3H virus has never been observed 
to disappear in C3H mice, but when intro- 
duced into RIII mice it can either remain ac- 
tive or disappear from them. 

Ultrastructure Cytology 

Dr. William Hall has his modern electron 
microscope laboratory set up and his research 
program underway. In studying the new strain 
DD with its premalignant, ormone responsive, 
plaques he has found that the malignant mam- 
mary tumors contain an abundance of both 
A and B mammary tumor virus particles, and 
the plaques also contain both A and B particles 
although not nearly so abundantly. In study- 
ing some of Dr. Evans' C3H mouse embryo 
cells in chemically defined media, he has ob- 
served many virus or virus-like particles which 
are as yet unclassified. Hepatomas in (C3HxY) 
Fj mice have not exhibited virus particles. 
They have, however, exhibited significant 
anomalies of the microbodies of the cells which 
represent the uricase component. Cells of spon- 
taneous and induced pulmonary tumors in our 
mice have failed to show any infectious vi- 
ruses with the E.M. 

Plasma Cell Tumors 

The plasma cell tumor is used extensively 
in the Laboratory by Potter and his group in 
studies on cell differentiation. Doctors Potter, 
Appella, and Mushinski have made consider- 
able progress in characterizing the gene-gene 
product relationships for the heavy chain locus 
in the mouse. In the mouse there have now 
been defined 7 immunoglobulin structural 
genes including 5 heavy chain genes: M, A, 



28 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



F, G, and H, and 2 light chain genes. A single 
plasma cell tumor utilizes only one heavy chain 
gene and one light chain gene, the others be- 
ing permanently "turned off". Structural var- 
iations resembling those in the light chains 
have been found in heavy chains. Iso-antisera 
have been produced that are specific for indi- 
vidual myeloma proteins and these provide val- 
uable tools in the search for antibody mole- 
cules that might resemble myeoloma proteins. 
Close linkage with no recombinants has been 
demonstrated between two heavy chain gene 
types. In the study of amino acid sequences 
the amino and carboxyl terminal sequences of 
five kappa light chains have been obtained. 
Structural analysis of the Fc fragments de- 
rived from mouse gamma and eta chains has 
shown the presence of twelve normal tryptic 
peptides indicating these genes are probably 
duplication products. This work has relevance 
to the understanding of the mechanism of the 
differentiation process and the mechanism of 
antibody formation. 

Mushinski is delving further into the pro- 
tein synthesis in these tumors using radio- 
active tracer techniques. He is investigating 
transfer RNA as the mediator of controlled 
amino acid ambiquities in these plasma cell 
tumors. 

Mclntire has been studying the plasma cell 
tumors and related reticulum cell tumors with 
more comparison to the multiple myeloma of 
man. In studying the macroglobulins of one 
tumor he observed the incorporation of a com- 
pletely different type of light chain into the 
immunoglobulin of the mouse, while formerly 
only one light chain had been shown. This is 
in line with human beings where two types 
of light chains have also been recognized. He 
has been studying renal disease in relation to 
the Bence Jones proteins produced by these 
tumors. Of 123 tested for Bence Jones pro- 
teins, 56 or 45 percent were positive, which 
is higher than the incidence cited for human 
beings. The kidneys associated with these tu- 
mors show cast formations in the convoluted 
tubules. Some reticulum cell neoplasms of the 
mouse other than plasma cell tumors produce 
these paraproteins and some of these are un- 
der study. 



Mclntire has studied plasma cell carcino- 
genesis in germ free mice with a minimum of 
antigenic stimulation, an undeveloped lym- 
phatic system, few plasma cells, and low level 
of immunoglobulins, and has observed in these 
mice a delay in plasma cell tumor develop- 
ment. Most of the tumors that arise are of 
other reticulum cell types. In this regard, thy- 
mectomy apparently has no influence on plas- 
ma cell carcinogenesis. 

Biochemical Genetics 

Hoffman has been studying the urinary pro- 
teins of the mouse by immunochemical meth- 
ods, agar gel double diffusion, and immuno- 
electrophoresis. Thirty-five inbred strains 
and substrains have been classified according 
to two different urinary protein types, Up-l a 
and Up-1\ In crosses between two prototype 
strains for type la (strain SWR) and type 
lb (strain C57BL/6) the F x shows a combi- 
nation of the two types and the F 2 segregates 
in a typical 1:2:1 ratio of the two parental 
types and the F a type. This indicates that 
these two urinary proteins are under the ge- 
netic control of a pair of co-dominant alleles 
at a single locus. Segregation ratios in first 
and second backcross generations have con- 
firmed this type of inheritance. By using sev- 
eral spearation techniques the la and the lb 
protein complexes have been isolated and 
purified. 

Hoffman has built up a linkage-testing 
stock of mice with 25 marker genes on 15 of 
the 20 chromosomes. With this stock he hopes 
to locate on specific chromosomes these genes 
for biochemical traits including low liver cata- 
lase, Ce, immunoglobulin-1, Ig-1, urinary 
protein, Up-1, and also a sparse coat texture 
gene, Ca De . 

Hoffman has also been identifying serum 
proteins of the rat. He has found that some 
of the rat serum proteins cross react with 
those of the mouse, whereas, others do not. 

All of this is directed towards building up 
a background of knowledge of biochemical 
genetics in the mouse that can later be related 
to the genetics of cancer in the mouse at the 
biochemical level. 



NATIONAL CANCER INSTITUTE 



29 



Genetics of Tumors 

Heston and collaborators have continued 
this study of effect of specific genes of the 
mouse on occurrence of tumors. The allelic 
genes lethal yellow, A y , and viable yellow, 
A Ty , have received greatest emphasis. Both 
increase occurrence of a number of tumors 
including mammary tumors, hepatomas, and 
lung tumors. 

It has been shown that the lethal yellow 
gene increases occurrence of mammary tumors 
independently of the mammary tumor virus, 
and studies of reciprocal transplantation of 
ovaries and of hypophyses show that its effect 
is not manifested through control of hormonal 
stimulation from either of these organs. It, 
therefore, appears that the gene is controlling 
the response of the mammary gland cell. 

In a study of hepatomas in reciprocal hy- 
brids between strain C3Hf and strain YBR 
with the A y gene it was observed that the A y 
gene, sex, and the strain of mother all could 
influence the occurrence of hepatomas, caus- 
ing the incidence to vary from zero in one group 
to 100 percent in one with intermediate inci- 
dences, depending upon the combination of 
these factors in the intermediate groups. How- 
ever, this was all closely correlated with the 
effect of these factors on normal growth. 

Progression of mammary tumors in mice is 
being studied through premalignant, hormone 
responsive, mammary plaques that occur in 
the unusual strain DD. The most significant 
observation on these plaques during the year 
is that whether or not they occur is dependent 
not only on the genotype of the mouse, but 
also on the strain of mammary tumor virus 
she carries. 

Hepatic Carcinogenesis 

Dr. Reuber has described the histology of 20 
hepatomas of the rat, including those of Dr. 
Morris that have been used extensively in stud- 
ies of enzyme activity. There were classified 
as (1) highly differentiated heptocellular car- 
cinoma; )2) well-differentiated hepatocellular 
carcinoma; (3) poorly differentiated or undif- 
ferentiated carcinoma; and (4) cholangiohep- 
atomas. He has classified an unusual neo- 



plasm of the liver that has been observed in 
(C3HxY)F! mice as of bile duct origin. 

In histogenic studies of the livers of ham- 
sters given N-2-fluorenylacetamide and N- 
2-fluorenyldiacetamide he has observed that 
cholangiofibrosis and cirrhosis preceded the 
development of well differentiated cholangio- 
carcinomas in both males and females. The 
lesions could be followed from (1) cholangio- 
fibrosis to (2) cholangiofibrosis with atypical 
cells to (3) small cholangiocarcinomas to (4) 
large cholangiocarcinomas with metastases 
to portoheptic lymph nodes. 

Dr. Reuber has described the development 
of carcinomas of the liver in rats following 
the administration of carbon tetrachloride. 
Although carbon tetrachloride has long been 
known to produce hepatomas in the mouse, it 
had not previously been observed to produce 
them in the rat. The older rats were more sus- 
ceptible to the induction of these tumors than 
were the younger rats, in contrast with hepa- 
toma induction with fluorenamine compounds 
where the younger rats are more susceptible. 
Older male rats also developed a higher inci- 
dence and more severe cirrhosis of the liver 
with administration of carbon tetrachloride 
than did the younger males. In the females 
the 12 and 24 week old animals had more 
severe cirrhosis than the 4 and 52 week old 
groups. 

The Malignant Transformation in Vitro 

Much of the effort of the Tissue Culture 
Section is directed toward the study of the 
malignant transformation in cells of various 
kinds in vitro, when and under what condi- 
tions it occurs, and what are the changes in 
the cell that can be associated with the trans- 
formation. 

One of the most interesting observations 
in this section is that of Evans and Andresen, 
later confirmed by Sanford, that the spontan- 
eous neoplastic conversion of C3Hf embryo 
cells in vitro is delayed with foetal calf serum 
as compared with horse serum. Chromosomes 
in neoplastic cells show structural rearrange- 
ments, but Sanford and Parshad have ob- 
served that the foetal calf serum appears to 



30 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



have a stabilizing influence on the chromo- 
somes. 

A number of changes in metabolic character- 
istics have been observed in the cells in vitro. 
Whether these are causally related to the ma- 
lignant transformation has not been ascer- 
tained. In some cases the changes may be 
merely owing to the cells growing in vitro. 

In one neoplastic strain Evans and cowork- 
ers have observed a change in metabolism of 
nucleic acid precursors. Some enzymes appear 
to be lacking or inactive. In another strain a 
change in lactate dehydrogenase behavior was 
noted that suggested a change in genetic con- 
trol rather than selection for cell type. 

In a line of cells on chemically defined me- 
dia studied by Westfall, lactate and malate 
dehydrogenase activities persisted at a high 
level but arginase and alkaline phosphatase 
showed changes in activity. In a line of liver 
cells arginase activity remained high, where- 
as there was a drop in production of urea, 
suggesting loss of critical liver function. The 
C3H mouse liver cells had twice the arginase 
activity as human skin cells in culture but 
both had high storage of glycogen. 

Sanford has observed in one clone of cells a 
great increase in glycolytic activity and at the 
same time an increase in tumor producing ca- 
pacity. Transformed lines showed very strik- 
ing histochemical variation in comparison 
with non-neoplastic lines. It is hoped to over- 
come this variation by cloning of the neo- 
plastic lines. In a study of a new strain of 
teratoma cells in culture that were diluted and 
inoculated into mice or culture it was found 
that even at the highest dilution the cells re- 
tained their differentiating capacity but the 
extent of differentiation varied inversely with 
the rate of tumor growth in vitro. With Dr. 
Rapp, Sanford has been studying Forssman 
antigen in hamster cells in vitro. Forssman 
antigen persisted in the cells for 12 months in 
vitro and since has been disappearing, al- 
though as yet the cells apparently have not 
become malignant. 

Sanford has demonstrated neoplastic trans- 
formation in hamster cells in vitro following 
treatment with polyoma virus. Conversion was 
not rapid and occurred at least one or two 



transplant generations following virus treat- 
ment. It appeared that transplantation anti- 
gens may be induced by the virus in cells 
already neoplastic. Morphologic alterations 
were noted but were not specific to virus in- 
fection. In looking for evidence of virus in her 
cell lines of both high- and low-tumor produc- 
ing capacity Sanford has found that some 
lines are positive for leukemia antigen. Dalton 
has found C particles in them but assays for 
biologic activity of the particles have always 
been negative. However, the neoplastic trans- 
formation may occur in cells free of the leu- 
kemia virus. 

Methodology in Tissue Culture 

Possibly the greatest advance in this area 
in recent years has been the development of 
the mass stirred fluid culture system with con- 
tinuous nutrient fluid renewal by Andresen, 
Bryant, and Evans. This system has been per- 
fected and now has been used for stain L cells 
in chemically defined media. Conceivably the 
cells could be maintained in this system indefi- 
nitely and examination of various aspects of 
growth could routinely be made through the 
malignant transformation. The system should 
also be of great value in the production of tis- 
sues for virus production. Commercial organ- 
izations will be very much interested in this 
system. 

Although the agitated fluid suspension cul- 
ture system developed in the Tissue Culture 
Section has been used for some time, Bryant 
and Evans continue to perfect it particularly 
in respect to growing cells in chemically de- 
fined media, and also to adapt and establish 
new lines of cells of various types including 
some mouse leukemia and mast cells, and a 
number of human cell lines. 

Five new lines of C3H mouse ambryonic fi- 
broblasts have been grown on the chemically 
defined media NCTC 135, demonstrating that 
this media is adequate for continuous growth 
of freshly explanted cultures. This, together 
with the fact that these cells can be main- 
tained for a long period of time, provides the 
system suitable for studying these cells through 
the malignant transformation and enhances 



NATIONAL CANCER INSTITUTE 



31 



the chances of causally relating observed 
changes to the transformation. 

A number of commercial establishments are 
now supplying media made according to the 
NCTC 135 formula and these are being tested 
for maintenance of cells in the Tissue Culture 
Section. Thus far none of these commercial 
media are wholly satisfactory, with the pos- 
sible exception of one powdered preparation 
of NCTC 135. 

Biochemistry 

Dr. Anderson has been summarizing her 
studies on glutamine antagonists and with Dr. 
Lyon has been continuing studies of mechan- 
isms of feedback control over "salvage" path- 
ways for pyrimidine nucleotide biosynthesis. 
Dr. Anderson and her program are transfer- 
ring to the Laboratory of Biochemistry that 
is expected to supply a better environment for 
her research. 

LABORATORY OF PATHOLOGY 

Introduction 

The work in Pathology is separated into two 
general areas, the Department of Pathologic 
Anatomy and the Laboratory of Pathology. 
The staff of Pathologic Anatomy performs the 
autopsies and examines the biopsies and sur- 
gical pathology and exfoliative cytology speci- 
mens for the Clinical Center. The annual re- 
ports on the number of these are attached. 

Another important operation maintained in 
the Laboratory of Pathology is the Patholog- 
ical Technology Section. A report (NCI 517) 
prepared by the head of this section, Mr. 
Joseph Albrecht, describes the work per- 
formed in this section. 

The primary aim of the staff of the Labora- 
tory of Pathology is to carry out experimental 
cancer research. For this a variety of tech- 
niques and experimental animals are used. 
Each senior staff member carries out his own 
experiments, but two or more members within 
the Laboratory may collaborate and individual 
members collaborate with many others in the 
National Cancer Institute, and in the National 
Institutes of Health. 



There is free exchange of information and 
assistance between and among all the pathol- 
ogists. While the Annual Report reflects in 
general the work of the two groups in pathol- 
ogy, it also shows that there is considerable 
interchange. Most of the members of the De- 
partment of Pathologic Anatomy have under 
study one or more problems involving cancer 
or some other disease in man which they carry 
out either independently or in collaboration 
with the clinicians. The autopsies and the sur- 
gical pathology specimens and biopsies are ex- 
amined with the greatest of care. They are 
initially studied by residents and their diag- 
noses are all reviewed by senior staff members. 

A notable achievement by the head of the 
Surgical Pathology and Postmortem Service of 
the Department of Pathologic Anatomy, Dr. 
Louis B. Thomas, is his work on a committee 
of the College of American Pathologists which 
has devised a nomenclature and code for 
pathological lesions. In collaboration with Dr. 
Arnold Pratt and the Biometrics Section of NIH 
this Systematized Nomenclature of Pathology 
is now being used in a computer retrieval sys- 
tem which permits the accurate and speedy 
retrieval of all autopsy and biopsy records in 
the National Cancer Institute. 

Most of the members of the Department of 
Pathologic Anatomy also carry out experi- 
mental research using animals, tissue culture 
and other special techniques. The residents in 
Pathologic Anatomy are particularly ambi- 
tious in carrying out research programs. 

Experimental Research 

The experimental work in the Laboratory of 
Pathology is not restricted to a single project, 
or to a group of closely related projects, but 
each pathologist follows his particular line of 
interest and training. Because of this diversity 
the projects in the Laboratory have been sep- 
arated into 6 rather loose categories for con- 
venience in preparing this summary. These 
are: 

Cancer in Man, and Related Animal Studies: 
Geographic pathology of cancer. 
Exfoliative cytology and cytogenetics. 



32 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Possible etiologic factors in human envir- 
onment. 
African lymphoma. 

Cancer in Animals: 
Induction and pathogenesis. 
Modifications in carcinogenesis. 
Biologic factors in neoplasia. 
Spontaneous tumors. 
Transplanted tumors. 

Viral Carcinogenesis and Related Problems 
in Virology: 
Polyoma virus. 
SV40 and adenovirus. 
Leukemogenic viruses. 
Virus replication. 
Interferon. 

Accumulation of Data on Laboratory Animals 

and Other Species: 
Information on laboratory animals. 
Phylogenetic aspects of neoplasia and 

comparative oncology. 

Development of New Techniques and Their 
Application. 

Collaborative Research: 
Methods. 
Examples. 

Cancer in Man, and Related Animal Studies 

Since nearly all the members of the Profes- 
sional Staff of the Laboratory of Pathology 
and Department of Pathologic Anatomy are 
medically trained, many research activities are 
correlated with the problem of human cancer. 
Several projects are directly utilizing human 
material, and others are testing in animals 
substances in the human environment that 
may be carcinogenic. 

Geographic Pathology of Cancer — In- 
formation is accumulating that certain forms 
of cancer in human population groups occur 
in a higher incidence than would be expected. 
Intensive study of such groups, and the recog- 
nition of possible etiologic agents which can be 
tested on experimental animals may identify 
some environmental factors which could be 
eliminated or controlled and thereby prevent 
many cases of human cancer. The following 
studies have this aim. 



Maps of different geographic areas are in 
preparation to show the relative frequency 
of different types of cancer. When it is recog- 
nized that some type is especially common, a 
more intensive study of the population group 
is indicated. (Dunham and Bailar) 

The following types of cancer are being in- 
vestigated in particular geographic area: 

Bladder cancer in New Orleans: White males 
over 60 in this city are frequently affected. All 
histologic sections have been reviewed by the 
same pathologists (Stewart and Rabson) to 
establish uniformity in diagnosis. Question- 
aires on past history and possible exposure to 
carcinogens are being analyzed, and a report 
is now in preparation. (Dunham) 

Uterine cancer in New York City, Israel 
and Washington, D.C.: This type of cancer 
is notably frequent in Negro women and in- 
frequent in Jewish women. Histologic sections 
have been examined from patients with cancer, 
and over 3,500 women interviewed. The data 
are being coded and analyzed in an effort to 
detect an environmental factor that may ac- 
count for the racial difference. (Stewart, Dun- 
ham, Thomas, Edgcomb) 

A study of cancer in North American In- 
dians has been started. Cancers of the uterine 
cervix and biliary tract, and basal cell car- 
cinoma of the skin, are frequent in this ethnic 
group. The histologic diagnoses have been made 
on 700 specimens in the NIAMD. Correlations 
with age and sex will be made. A survey of 
the literature revealed that very little reliable 
information is now available on the Indians. 
(Dunham, Laqueur) 

Lung cancer in veterans of World War I: 
The medical history and biopsy and autopsy 
material from this group are gathered. All 
histological material has been reviewed by the 
same pathologist, and a uniform classification 
of histologic types has been made. Different 
histologic groups may have different signifi- 
cance and relationship to environmental fac- 
tors. This survey indicated the unreliability 
of random histologic diagnoses which have 
often been accepted uncritically by statis- 
ticians or epidemiologists. The results have 
been coded and a manuscript is in preparation. 
This study will supply important information 



NATIONAL CANCER INSTITUTE 



33 



on the influence of smoking on the incidence 
of lung cancer within this group. (Herrold) 

Gastric cancer in Japan: Cancer of the stom- 
ach is remarkably frequent in several unrelated 
human populations, and in Japan, this type of 
cancer is especially common. Migrant Japa- 
nese in Hawaii offer a good group of genetic- 
ally similar people in a different geographic 
area for comparison. The histologic sections 
are being reviewed by the same pathologist 
(Herrold), and any association with gastric 
ulcer, polyps or intestinal metaplasia is noted. 
A correlation will be made with data collected 
by a team of epidemiologists. An investigation 
is also made for viruses (Bryan); and elec- 
tron microscopy is done when possible. His- 
tochemical studies on the human material in- 
dicate a change in gastric mucins, and corre- 
lated studies of Syrian hamsters support this 
finding. This is a long term study that will re- 
quire many years, but a preliminary report 
will be given at the International Congress in 
Japan in 1966 (Herrold, Stewart, pathologists 
from Japan). 

EXFOLIATINE CYTOLOGY AND CYTOGENETIC. — 

A diagnostic service in exfoliative cytology is 
supplied to the Clinical Center, but in addi- 
tion to this, a number of research studies are 
in progress in collaboration with various clin- 
ical services. These relate to the presence of 
neoplastic or other cells on body surfaces or 
in fluids, and sex chromatin in exfoliated cells 
(Malmgren) . It has been determined: 

Number of neoplastic cells in the blood of 
cancer patients is not closely correlated with 
metastasis. 

Cancer cells are often recovered from wound 
washings after cancer surgery, but time will 
be required to determine whether the fre- 
quency is correlated with recurrence in the 
wound site. 

Leukemic cells in the spinal fluid provide a 
reliable indication of central nervous system 
involvement and in the assessment of therapy. 
Evaluation of intrathecal treatment can be 
made by observation of neoplastic cells in 
spinal fluid. 

An improved technique using a milipore fil- 
ter makes the identification of sex-chromatin 
in cells from the buccal mucosa more reliable. 



A cell line from a primary lymphoma of 
the ovary in an American woman has been 
studied using cytogenetic techniques. Ninety 
percent of cells in the primary tumor carried 
a marker chromosome which was not seen in 
the established continuous tissue culture line. 
This indicated that the marker chromosome 
was not an inseparable and necessary feature 
of the malignant cell. (Chu) 

A system for coding findings in exfoliative 
cytology has been prepared, and is now in 
operation. (Malmgren) 

Possible Etiologic Factors in Human 
Environment. — An important area in cancer 
research is the identification of environmental 
factors in a human population that may alter 
the expected incidence of cancer. Notable prog- 
ress in the prevention of cancer has come from 
recognition of the carcinogeni activity of 
soot, shale-oil, and radioactivity. Once an en- 
vironmental factor is suspected, tests on ani- 
mals are necessary to prove that it is carcino- 
genic and in a complex substance it is impor- 
tant to identify the most potent fraction. A 
number of experiments with this aim are now 
in progress in the Laboratory of Pathology. 

Absorbates from drinking water from New 
Orleans, where bladder cancer is high, and 
Birmingham, where it is low, are concentrated 
and given to mice in alcohol or chloroform. 
Lung tumors, a delicate indicator of carin- 
ogenicity were equally frequent in control and 
experimental groups. (Dunham) 

Betel quid chewing is associated with oral 
cancer in many areas of the world. Ingredi- 
ents of the quid have been tested in the hams- 
ter cheek pouch. Calcium hydroxide caused the 
most damage to the mucosa, and produced in- 
flammatory and hyperplastic lesions and epi- 
thelial atypia, but no cancer. Other sub- 
stances were ineffective. A paper describing 
these results has been accepted for publica- 
tion in the British Journal of Cancer. (Dun- 
ham) 

The increasing number of lung cancers in 
man is a cause for concern. No exact prototype 
of human lung cancer is known in animals, 
but a technique has been devised where a 
substance for testing can be incorporated in 
a beeswax pellet, and inserted into the lung 



34 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tissue of a rat. When cancer develops it is 
epidermoid as in human lung - cancer. The for- 
mation of keratinizing cysts in the rat indi- 
cated a potentially cascinogenic substance. 
This technique will make the investigation of 
potentially carcinogenic substances for man 
more accurate. (Stanton) 

Attempts to produce bladder cancer in 
hamsters with schistosoma were unsuccessful, 
but since the Egyptian and the Gold Coast 
strain were each used, a comparison and search 
for histologic differences in the effects can be 
made. (Thomas) 

DMSO, a solvent suggested for therapy in 
man was tested in mice. No increase 
in tumors was noted. (Dunham) 

The long term effects of enovid (the anti- 
fertility pill) are under investigation in mice. 
It was found that the sterility dose is 4 or 
more times that required for women. All ani- 
mals are living after a year. One mouse that 
received enovid when newborn developed a 
granular cell myoblastoma of the cervix. Sim- 
ilar tumors were found in mice receiving es- 
trogens when newborn. (Dunn) 

Numerous other substances to which human 
beings are exposed have been tested, or are 
now being tested. Dihydrosafrol (DHSF) a 
food coloring proved to be toxic to OM rats 
on the usual laboratory chow, but when on an 
80% corn meal diet the rats lived longer and 
some atypia of the esophageal mucosa was 
found. Esophageal cancer is high in Curacao, 
and natives use many decoctions of plants. 
Decoctions from 12 plants used by 11 patients 
with esophageal cancer have been given to ro- 
dents but it is too early to expect results. The 
quantity of zinc and copper in the soil has 
been correlated with the occurrence of certain 
human tumors. To test this in animals, squares 
of zinc, copper or iron were embedded in sub- 
cutaneous tissues of rats. Substances from 
heated fats were given to rats. Experiments 
begun by Dr. Wilhelm Hueper on the car- 
cinogenicity of heated fats are being con- 
tinued. The results are incomplete, but there 
is an indication of a weak carcinogenic ac- 
tion that is enhanced by repeated heating of 
the fat. (O'Gara) 



African Lymphoma. — The frequency of 
this form of cancer in children in Africa has 
aroused much speculation as to possible etio- 
logic factors. A series of experiments have 
been conducted in the Laboratory of Pathol- 
ogy, and comparisons made between a lym- 
phoma in an African child, and a tumor of 
similar cytology in an American woman. Each 
of the tumors has been grown in tissue cul- 
ture. Cells are similar and resemble trans- 
formed lymphocytes. In vitro studies of the 
cell line from the African lymphoma showed 
40-77 chromosomes. Interferon was found in 
supernatant fluid. The cell line from the 
American woman produced immunoglobulins 
(Rabson). Herpes-like particles have been seen 
in both lymphoma lines and attempts to isolate 
a virus and to clarify the role of the particles 
in these tumors continue. The tissue culture 
cells could be infected with the herpes virus, 
but other viruses so far tried have not pro- 
liferated in the cultures. Twenty-one malig- 
nant lymphomas of various histologic types ob- 
tained from Clinical Center patients failed to 
show viruses or virus-like particles by elec- 
tron microscopy. (O'Conor) 

Cancer in Animals 

Many of the studies of cancer in animals are 
concerned with (a) induction and pathogene- 
sis, or the steps by which cancer develops 
after exposure to a carcinogen; (b) modifica- 
tions in the activity of carcinogens produced 
by altered environmental conditions or sub- 
stances; (c) biologic factors of neoplasia such 
as the behavior of tumors in tissue culture, 
and transfer by vectors; (d) spontaneous and 
unusual tumors; and (e) transplanted tumors. 

Introduction and Pathogenesis. — Devel- 
opment of gastric and skin appendage tumors 
produced by 2-7-FAA when given ,to preg- 
nant or lactating mothers, to newborns, and 
to adult rats is being studied. The distribution 
of skin appendage tumors produced by this 
chemical is being plotted on maps of the skin 
surface and a paper on histogenesis has been 
prepared. The tumor yield in the offspring is 
increased if 2-7-FAA is administered during 
both pregnancy and lactation. Carcinogenic 



NATIONAL CANCER INSTITUTE 



35 



action is less certain if given during- preg- 
nancy or lactation only. (Stewart, Snell) 

Monkeys were injected with MCA in the 
wall of the stomach 15-17 years ago. No can- 
cers have been found, but degenerative 
diseases such as arthritis have appeared. 
(Stewart, Snell) 

Several studies have been done with the ni- 
trosamines which have been found to be potent 
carcinogens. Nitrosamine compounds given by 
a variety of routes in hamsters produced tu- 
mors at similar sites, indicating that the dis- 
tribution, metabolic pathways, and excretion 
of the carcinogen were the critical factors. 
Tumors of the olfactory neuroepithelium in 
Syrian hamsters were of special interest, be- 
cause in previous reports these may have been 
mistaken for brain tumors by others. These 
studies emphasize that carcinogens may reach 
remote sites, and that when the site of tumor 
formation is in the lung, the carcinogen did 
not necessarily reach the lung by inhalation. 
(Herrold) 

Methylnitrosourea (MNU) was given by a 
number of routes to rats. Tumors histologically 
resembling neurilemmomas appeared at many 
different sites. Since use of this substance as 
a solvent has been proposed, awareness of its 
carcinogenic potency is important. (Stewart, 
Snell) 

Nitrosamine was given orally in various ve- 
hicles to general-purpose mice. Papillomas of 
the esophagus and forestomach were found in 
a few. (O'Gara) 

Syrian hamsters were given benzopyrene by 
intratracheal instillation and early changes in 
the bronchial epithelium were described. No 
tumors resulted from atmospheric pollutants 
or tobacco tar. An unanticipated finding from 
this experiment was the development of cryp- 
tococcus neoformans meningitis in one ham- 
ster. This disease occurs in man, where it has 
been presumed that the organism is blood- 
borne from a focus in the lung. Observations 
with the hamster suggest that the primary 
focus may be the nasal cavity and sinuses, 
from which sites the infection extends to the 
meninges. (Herrold) 

A prototype of human osteogenic sarcoma 
has been produced in rats by copper chelated 



N-OHAAF, introduced into the medullary 
cavity. Following this observation new experi- 
ments have been started. An improved tech- 
nique is used in inbred rats so that any tu- 
mors that develop can be transplanted. (Stan- 
ton) 

Hepatic cancer is being produced success- 
fully in primates after a short induction per- 
iod. Newborn and infant monkeys were used, 
and 13 hepatic neoplasms were produced by 
DENA. Successful intracerebral transplants 
have been made. This is the first time quick 
induction of cancer and transplantation has 
been accomplished in primates. The trans- 
planted tumors have now been used in thera- 
peutic trials. (O'Gara, Kelly) 

Chronic myelogenous leukemia developed in 
one monkey that received MIH, a drug used 
for treating Hodgkin's disease. Although leu- 
kemia has been induced in one monkey by 
irradiation this is the first time it has been 
induced in a primate by a chemical. (O'Gara, 
Kelly) 

Modifications in Carcinogenesis. — Vita- 
min A delayed the appearance of tumors of 
the forestomach in hamsters given DMBA. No 
inhibition of skin cancer was noted. (Chu, 
Malmgren) 

Hepatocarcinogenesis in the rat has been al- 
tered by dietary modifications: (a) Three 
times as much carcinogen N,N-Dimethyl-^>- 
phenylazoaniline is required to induce hepa- 
tomas on a complete diet, as it takes to induce 
cancer in rats on a deficient semi-synthetic 
diet. (6) n a complete Purina laboratory 
chow diet, the carcinogen N,N-Dimethyl-p- 
(m-toiylazo) aniline induced cysts and cho- 
lageofibrosis and a rare hepatoma, but the same 
dose of carcinogen gave 100% incidence of 
hepatoma on a deficient semi-synthetic diet, 
(c) An extract of U.V. irradiated fat in con- 
junction with subcarcinogenic dose of N,N- 
Dimethyl-p-phenylazoaniline had a tendency 
to induce hepatomas. The extract alone caused 
considerable liver damage but no neoplasms. 
(Mulay) 

Manipulation of copper and zinc concentra- 
tion in the rat diet failed to induce neoplastic 
lesions. Thus it fails to support the contention 
that a higher incidence of gastric cancer, in 



36 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



some areas, is related to use of food grown 
exclusively on soils with copper and zinc im- 
balance. (Mulay) 

Early alterations preceding the develop- 
ment of cancer are being investigated. 

Rats fed a hepatocarcinogenic diet showed 
a rise in adrenal steroid concentration, two 
weeks after the start of the diet and con- 
tinued high. The significance of this finding 
to azo dye carcinogenesis is discussed. (Mulay) 

Biologic Factors in Neoplasia. — Cell cul- 
tures have been made from a liver tumor in 
a rhesus monkey (see O'Gara) above. The cells 
grow slowly and the morphology remains 
epithelial with some attempt at differentiation, 
but show no evidence of function. Transplants 
back to the monkey brain have been successful. 
(Dawe) 

The ability of insect vectors to transmit 
neoplasms is under investigation. A reticulum 
cell sarcoma in the hamster has been under 
observation for several years. It was proved 
that it could be transmitted by feeding tumor 
tissue to susceptible hamsters and it has now 
been shown that it can be transmitted by the 
bite of the Aedes mosquito. Transmission is 
by cells. No multiplication of the cell or a virus 
within the mosquito could be noted. Attempts 
to transmit Rauscher cells or the virus have 
failed. Transfer of Moloney virus by the mos- 
quito was successful in 2 of 61 trials. (Ban- 
field) 

Spontaneous Tumors. — These are always 
under study in the Laboratory of Pathology 
and descriptions and classifications of them 
are published at intervals. A considerable 
number of histologic sections of retriculum 
cell sarcomas have been accumulated by Dr. 
Margaret Deringer. Since this group of tumors 
is especially confusing and complex the slides 
are being reviewed and a publication on the 
histological features is being prepared. (Dunn) 

Transplanted Tumors. — Observations on 
transplanted tumors and their behavior fur- 
nish valuable information on neoplasia. A 
number of these are carried and studied in 
the Laboratory of Pathology, and are made 
available to other investigators. Among many 
tumors of interest are an adrenal cortical tu- 
mor, gastric adenocarcinomas, a neurilem- 



moma, and a mesothelioma (Stewart and 
Snell). Others carried in mice are a myoepith- 
elioma with a leukemoid reaction, a kidney 
tumor, and a granular cell myoblastoma. 
(Dunn) 

Viral Carcinogenesis, and Problems of 
Immunology and Resistance 

The same interest in pathogenesis and the 
mechanisms of carcinogenesis will be found 
in the experiments carried out by pathologists 
with viruses as was noted in experiments with 
chemical carcinogens. Experiments now in 
progress are concerned with: Polyoma virus, 
SV40 and adenovirus 12, leukemogenic viruses, 
and Interferon. 

Polyoma Virus. — Studies on the interac- 
tion of mesenchymal and epithelial elements 
in organ culture, and the effect of the polyoma 
virus have continued. The specificity of the 
mesenchyme for the epithelium is critical cr-1 
some combinations produce no neoplastic 
change, even in the presence of PV. Continued 
contact with natural mesenchymal tissue is re- 
quired for epithelial neoplasia, and neoplasia 
will not develop until some morphogenesis and 
differentiation take place. The salivary gland 
rudiments must reach a certain stage before 
neoplasia results. Viral oncogenesis is not sim- 
ply the result of interaction of the viral ge- 
nome with the cell genome — epigenetic fac- 
tors also operate and a proliferative stage is 
required. (Dawe) 

The behavior of a polyoma induced tumor 
in the hamster was compared with a spon- 
taneous and a carcinogen-induced tumor after 
intravenous injection of dissociated cells. The 
resulting nodules in the lung after injection of 
the polyoma tumor were interpreted as in- 
flammatory reactions rather than true neo- 
plasms. (Herrold) 

Thymectomy was shown to increase the on- 
cogenic effect of the polyoma virus. Of par- 
ticular interest was the observation that 
C57BL mice resistant to polyoma oncogenesis 
were made susceptible by thymectomy but sen- 
sitized spleen cells will inhibit this suscep- 
tibility even when given many weeks after the 
viral infection and thymectomy. A morphologic 



NATIONAL CANCER INSTITUTE 



37 



study of this effect is now in progress. (Stan- 
ton, Law) 

Two strains of the polyoma virus with dif- 
ferent oncogenic potentials but with a similar 
capacity for the induction of tumor antigens 
are available. These have been tested by the 
tumor rejection assay. Paradoxically, the more 
oncogenic virus inhibited the ability of the 
mouse to reject a non-polyoma tumor, possibly 
because of thymotrophic action. (Friedman) 

Sv40 and Adenovirus. — (a.) Newborn ham- 
sters were infected with SV40 and adeno 
12 simultaneously and the progeny of the two 
viruses grown in mixed infection. The SV40 
had previously been shown to induce tumors 
with large cells, while the adeno 12 tumor 
cells were small and hyperchromatic. The tu- 
mors that developed early were of a histologic 
type characteristic of adeno 12; tumors that 
developed later were of SV40 type, (b) A 
"T" antigen was shown by immunofluores- 
cence to be correlated with stippling seen by 
electron microscopy in monkey kidney cells 
with adenovirus 12. (c) The hybrid virus 
adeno 7-SV40 designated E46 produced SV40 
type tumors, described as papillary ependy- 
omas when given intracerebrally. When given 
subcutaneously, it produced "mixed" tumors 
showing histologic areas like SV40 and other 
areas like adeno 12. (Rabson) 

Electron microscopic studies on mixed viral 
infections are continuing. Simultaneous repli- 
cation within the same nucleus of SV40 and 
herpes simplex virus has been demonstrated 
in green monkey kidney cells. Hybrid E46 has 
protein crystalline structures in the nucleus 
which are not found in the parent adeno 7 or 
SV40. EM studies on arbovirus infection are 
being correlated with biochemical studies in 
chicken fibroblasts infected with Semliki forest 
virus. (O'Conor) 

Leukemogenic Viruses. — Moloney virus in- 
fection in rats has disclosed previously in- 
apparent Bartonella organisms in the blood 
and encephalazoon lesions in the brain in the 
preleukemic state. This indicates an alteration 
in the defense mechanism during the preleu- 
kemic state, and raises the question whether 
an atypical response to infection may be found 



in a preleukemic state in man, and clinicians 
should be alert to this possibility. (Stanton) 

BALB/c mice infected with Rauscher virus 
and malaria die earlier. Malaria infection also 
causes an increase in macroglobulin. Changes 
in the globulins might be of diagnostic aid in 
malaria and leukemia. (Edgcomb) 

BALB/c mice with Rauscher virus devel- 
oped an extreme erythroblastic reaction but 
no leukemia. The erythroblastic reaction was 
inhibited (but not prevented) by feeding pro- 
phylthiouracil or by repeated blood trans- 
fusions (Dunn, Malmgren). Moloney virus in- 
fection was enhanced by DNA and RNA ob- 
tained from a commercial source. (Malmgren) 

Virus Replication. — Replication of an 
RNA virus is under study. A model cytopathic 
RNA virus infection with Semliki Forest 
Virus (arbovirus, group A) is used as a model 
in order to elucidate the mechanism of repli- 
cation of RNA viruses including possibly leu- 
kemia viruses. Studies have shown that in 
addition to the single standard RNA present 
in infectious virus, 2 other replicative forms 
are present in infected chick cells. One appears 
to be a double stranded viral forms. These 
forms are present in 3 distinct cytoplasmic 
particles. Protein synthesis by virus and the 
interrelationship between the 3 cytoplasmic 
particles are being investigated. 

Interferon. — Mechanisms of interaction of 
interferon and the possible role in carcino- 
genesis are under study. The action involves 
action protein synthesis. Interferon appears to 
limit production of viral DNA in vaccinia 
virus. (Friedman) 

Accumulation of Data on Laboratory Animals 
and Other Species 

Because the training of pathologists is not 
restricted to one organ system or type of dis- 
ease, they are often able to contribute to the 
general knowledge of the normal and patho- 
logic anatomy of many different animal spe- 
cies. This contribution is often incidental to 
the main purpose of cancer research, yet it is 
indispensable to the intelligent use of biologic 
material, because, unless the normal anatomy 
and spontaneous pathologic alterations are 



38 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



recognized, it is impossible to interpret the 
effects of experimental procedures. 

For ten years or more autopsies have been 
performed on old rats of 6 inbred strains. The 
incidence of tumors and other lesions has 
been accurately recorded. This information 
has become increasingly more complete and 
valuable and facts derived from this survey 
are often requested by pathologists within the 
NIH and from outside. A detailed review of 
renal lesions in these old rats, with a review 
of the literature was recently presented at a 
Conference on Spontaneous Diseases of Rats 
and Mice sponsored by the Nuffield Foundation 
in England. (Snell, Stewart) 

Mastomys have been introduced as a new 
laboratory species, and information on the nor- 
mal anatomy and spontaneous diseases is being 
accumulated by autopsies performed on 200 
animals from the colony maintained at the 
NCI. These animals lived out the normal life- 
span. Tumors were frequent in the glandular 
stomach, the liver and the thymus. A kidney 
lesion resembling human glomerulonephritis 
was frequent. The female has a well developed 
prostate gland. (Stewart, Snell) 

Another species that has been used exten- 
sively as a laboratory animal for a decade or 
more is the hamster, yet comprehensive and 
reliable information on the normal anatomy 
and spontaneous disease of this useful rodent 
is still lacking. References describing the nor- 
mal anatomy are now being accumulated and 
information on the normal anatomy and spon- 
taneous diseases in hamsters used as controls 
in a variety of experiments is being compiled. 
(Herrold) 

Information from personal observations and 
the literature is being accumulated on the nor- 
mal and pathologic anatomy of the laboratory 
mouse. A paper on renal disease in mice and 
on amyloidosis in mice was given at the recent 
Conference in England. (Dunn) 

Complete autopsies are being performed on 
both control and experimental monkeys. At the 
present time information on the normal and 
pathologic anatomy of monkeys is deficient, 
scattered, and inaccurate, and reliable knowl- 
edge will be supplied only by competent pa- 



thologists who perform many autopsies. A 
form of vascular disease resulting from subcu- 
taneous injections of polycyclic hydrocarbons 
as been described. (O'Gara) 

An International Conference on Lung Tumor 
in Animals was held in Perugia, Italy, in June 
1965. A principal address was delivered on 
comparison of histologic lung tumor types in 
fowls, lower animals and man. In preparation 
for this address, all previous descriptions on 
lung tumors in these animal species were re- 
viewed, and the histologic sections were exam- 
ined when available. Many of these came from 
the Philadelphia Zoo. This critical review re- 
vealed that many tumors previously accepted 
as primary were probably metastatic. Since 
captive wild animals and domestic are closely 
associated with man and share his environ- 
ment, any neoplasms found have special signi- 
ficance. (Stewart) 

Phylogenetic Aspects or Neoplasia. — 
An important project has been started on 
phylogenetic aspects of neoplasia in coopera- 
tion with the Smithsonian Institution and ma- 
rine biological laboratories. The collection and 
identification of neoplasms in invertebrates 
has been started, a literature survey now hav- 
ing over 300 entries will be continued and a 
registry of specimens will be made. Neoplasia 
in planaria and cockroaches is being investi- 
gated under laboratory conditions. The pla- 
naria have developed a curious spontaneous le- 
sion with inclusion bodies, but it is doubtfully 
neoplastic. Soft shell clams are being investi- 
gated for neoplastic changes. It is apparent that 
adequate criteria have not been used previ- 
ously to determine neoplasia in lower forms. 
(Dawe) 

Studies in comparative oncology include 
an effort to determine the neoplastic response 
of fish to chemical carcinogens. Cycasin was ad- 
ministered and severe liver damage produced 
which the fish survived. Hepatic tumors de- 
veloped in a restricted interval of time from 
12-15 weeks. This fact together with the small 
size of the liver which permits serial section- 
ing should make this species a valuable indi- 
cator of potential carcinogenicity. (Stanton) 



NATIONAL CANCER INSTITUTE 



39 



Development of New Techniques and Their 
Application 

Since modern pathology is not static and is 
not restricted to anatomical dissection and ob- 
servations with the light microscope, new tech- 
niques must be introduced and old ones im- 
proved. New electron microscopic techniques are 
being developed especially. 

The electron microscope probe and the scan- 
ning electron microscope offer greater preci- 
sion in morphologic studies. Biological appli- 
cation of these new instruments will first be 
on an experimental basis. It is now possible to 
make a determination of the amount of phos- 
phorus in the nucleus. Three dimensional per- 
spective should become possible, and a better 
knowledge of the cell surface can be obtained. 
An analysis of tissue in situ can be accom- 
plished by these new methods. (Banfield) 

A new technique, the Transer, eliminates 
fixation and embedding of tissues for EM ex- 
amination, and offers new opportunities for 
analytical histological investigation. This will 
be especially valuable in continuing studies on 
collagenous connective tissue and elastin. 
(Banfield) 

Ferritin conjugated antibody studies are be- 
ing applied in viral oncology. An effort is be- 
ing made to improve the specificity of present 
methods while preserving cell ultrastructure. 
Findings are being correlated with immuno- 
fluorescence techniques. (O'Conor) 

Mice have been placed on a complete liquid 
diet, the commercially available Metrecal, and 
found to be healthy after 6 months. Develop- 
ment of an appropriate basic liquid food for 
mice would be desirable in many experiments, 
and possibly even for routine matintenance. 
(Dunn) 

Collaborative Research 

It is recognized that many research projects 
at the National Cancer Institute require the col- 
laboration of a pathologist, especially in the 
final evaluation of the effect of an experimen- 
tal procedure on laboratory animals. The Lab- 
oratory of Pathology has always tried to make 
this assistance available. 



Methods. — The experimental pathologist 
may take an active part in planning an ex- 
periment and in following it through; he may 
take the responsibility for all autopsies and 
histologic diagnoses in an experiment; he may 
review only the histologic sections in a given 
experiment; or may serve as a consultant to 
review selected material with no responsibility 
for the entire experiment and its publication. 
Finally, he may make use of material accu- 
mulated by other investigators for indepen- 
dent studies concerning pathologic alterations. 
It is emphasized that full collaboration of the 
pathologist at the time the experiment is 
planned is the most satisfactory arrangement 
for it insures the best and most economical se- 
lection of material for pathologic studies. 

The impressive amount of collaborative work 
carried on by the staff in Pathologic Anatomy 
with clinicians at the National Institutes of 
Health is shown in the report submitted by 
Dr. Louis Thomas (NCI-853). Pathologists also 
collaborate in experimental work on clinical 
problems which may not be directly related to 
cancer, but which could not be accomplished 
without pathology assistance to physicians of 
the Clinical Center staff. 

In addition to the use of the light micro- 
scope and standard autopsy procedures, indi- 
vidual members of the Laboratory of Pathology 
have become proficient in special techniques 
such as fluorescent antibody visualization, elec- 
tron microscopy, tissue culture, autoradiog- 
raphy, exfoliative cytology, special cytology, 
and histochemistry. These special skills are 
often made available in collaborative studies. 

Example:. — It would be tedious to consider 
all the collaborative work now in progress in 
the Laboratory of Pathology, and this should be 
unnecessary since it is covered in reports from 
other laboratories. However the following are 
noteworthy: 

With other scientists in cancer research: 
(a) Tumor cells were transplanted to day-old 
hamsters and the subsequent tumors were 
were treated with clam juice. Macrosis and 
regression was produced in the small tumors 
(Chu, C. P. Lee), (b) Variations in the estrone 
cycle were produced by continuous light and 
by interruption of the pathway to the pineal 



40 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



glad in rats (Chu, Wurtman). (c) Degenera- 
tive joint disease of Mastomys has been stud- 
ied (Snell, Sokoloff). (d) The histology of an- 
terior chamber transplants from tissue culture 
cells made to the eyes of mice has been de- 
scribed (Dunn, Evans). 

Research on clinical problems: (a) Emphy- 
sema is a crippling disease of increasing fre- 
quency. A probable factor in its pathogenesis 
is increased air pressure in airways. This has 
been produced by experimental procedures in 
rabbits. The amount of emphysema and tis- 
sue damage correlated with the air pressure, 
but spontaneous healing occurred, (b) Myo- 
cardial mechanics of the dog's heart were 
studied during systole. Apparently two groups 
of differently oriented muscle fibers determine 
dimensional changes during systole, (c) A se- 
ries of renal biopsies in lupus nephritis was 
studied in order to determine how uniform and 
widespread the lesions might be. (d) Endo- 
metrial changes and ovulation in non-fertile 
women after treatment with gonadotrophic 
hormones were studied. Induction of ovulation 
was produced in some cases. (Powell and 
others) 

Use of special techniques: Fluorescent anti- 
body studies. The following are some now in 
progress: antigens in patients with mycosis 
fungoides, antigens in human leukemia cells, 
monkey kidney cells with adena 12 and SV 40 
virus, PV tumor antigen in lytic infection of 
mouse embryo cells, antigens to adenovirus 7- 
SV40 in induced neoplasms of the hamster kid- 
ney. (Malmgren and others) 

Electron microscopy of normal and patho- 
logic tissue. Application to epidemic diarrhea 
in mice, where virus particle so far have been 
found only in the lumen. (Banfield and others) 

LABORATORY OF PHYSIOLOGY 
Cancer Physiology Section 

Dr. S. H. Wollman and research associate 
Dr. J. E. Loewenstein are continuing their 
efforts in studying the iodide-concentrating 
mechanism of the thyroid gland. This investi- 
gation is concerned with (a) the mechanism 
by which the thyroid gland maintains a con- 
centration of iodide elevated above that in the 



blood, and (b) the mechanism by which the 
thyroid gland accumulates protein-bound io- 
dide. Kinetic studies on the exchange of radio- 
iodide between individual thyroid follicles and 
blood have indicated that their kinetic proper- 
ties are dependent on the size of the follicle. 
Experimental results support previous theoret- 
ical considerations made by Wollman and Dr. 
G. Andres (former research associate) that the 
average epithelial cell clears radioiodide from 
the blood and releases concentrated radioio- 
dide back to the blood to the same extent in 
follicles of all sizes and that the radioiodide 
transport properties of follicles depend on their 
surface to volume ratio. Kinetics of equilib- 
rium labeling of the protein-bound iodine in 
individual follicles reveal that at equilibrium 
the concentration of radioiodine in follicles, 
and ^therefore the stable iodine, is independent 
of follicle size. Follicles at the periphery of the 
thyroid lobe equilibrate slower than do the 
central follicles. The rate of equilibrium of 
central follicles (which parallels the rate of 
release of organic radioiodine) varies in- 
versely as follicle size. 

Dr. Rabinovitz and Dr. Honig and research 
associates Drs. Waxman and Freedman have 
have been doing extensive work on protein syn- 
thesis and its control in normal and tumor 
cells. This entails studies on the pathway (s) 
taken by amino acids in the synthesis of pro- 
tein and the controls exercised by the cells in 
regulating these processes. As information is 
obtained concerning protein synthesis in the 
normal cell, it is applied to the tumor-bearing 
animal with the view of selective inhibition of 
protein synthesis in cancer cells. With Dr. 
Honig, studies have been made of the require- 
ment of ribonucleic acid synthesis for some 
respiration dependent biosynthetic pathways 
in Sarcoma 37 ascites cells. Inhibitors of RNA 
synthesis in Sarcoma 37 cells, such as actino- 
mycin D, also inhibit the synthesis of proteins 
and sterols in these cells. They have found that 
the inhibition of the synthesis of protein and 
sterols can both be prevented and relieved by 
glucose or other glycolyzable substrates. The 
inhibition of protein synthesis has been dem- 
onstrated to occur at the step involving amino- 
acyl-transfer RNA formation. The level and ac- 



NATIONAL CANCER INSTITUTE 



41 



tivity of the enzyme catalyzing this reaction 
was identical in cells treated and not treated 
with actinomycin D. There appeared to be no 
difference in ATP level or in rate of ATP syn- 
thesis in cells with impaired biosynthetic ca- 
pabilities. They believe that there is present a 
short-lived RNA which is involved in the avail- 
ability of oxidative energy for biosynthetic 
processes. With Dr. Waxman, studies on the 
control of reticulocyte polyribosome content and 
hemoglobin synthesis by heme indicated that 
hemin enhances polyribosome stabilization and 
formation as well as globin synthesis. This ef- 
fect of hemin duplicates the results previously 
reported for iron salts. Cobaltous ion, deutero- 
hemin and zinc protoporphyrin were all capa- 
ble of producing the enhancing effects on poly- 
ribosomes and globin production. Nickel and 
zinc salts, as well as other cations, were in- 
effective. Lead salts, which lead to reticulocyte 
polysome disaggregation, apparently produce 
this effect by the inhibition of iron incorpora- 
tion into protoporphyrin. These data suggest 
that hemin is an intracellular mediator of reti- 
culocyte polyribosome assembly and maintains 
the functional integrity of the biosynthetic 
apparatus in these cells and precludes the for- 
mation of globin under conditions of iron de- 
ficiency. With Drs. Freedman and Honig, stud- 
ies on the control of nuclear-histone synthesis 
in chicken reticulocytes indicate that these re- 
ticulocytes in vitro can synthesize hemoglobin, 
nuclear ribonucleic acid and nuclear proteins 
including histones. If one adds actinomycin 
midazole, inhibition of nuclear ribonucleic acid 
D or 5, 6-dichloro-l-/3-D-ribofuransyl-benzi- 
synthesis occurs promptly. This also led to a 
rapid decrease in the rate of synthesis of cer- 
tain nuclear proteins, particularly histones. 
One histone fraction characterized by its solu- 
bility in 5% and insolubility in 20% trichloro- 
acetic acid was particularly sensitive to inhib- 
itors of RNA synthesis. These results suggest 
that the synthesis of specific histones may be 
under precise control in the reticulocyte nu- 
cleus through the mediation of newly-formed 
ribonucleic acid. 

Dr. Reid continues his studies on the deter- 
mination of the pattern of excretion of a broad 
group of metabolites by leukemic patients with 



special reference to nucleic acid congeners. 
The techniques involved are both chemical 
and mathematical. By employing chemical 
methods, urinary specimens are fractionated 
into a large group of mixed fractions of nu- 
cleic acid congeners. Mathematical techniques, 
primarily linear algebra, are employed to fur- 
ther resolve these mixed fractions by means of 
computational analysis of their ultraviolet ab- 
sorption spectra. Data-processing equipment 
is used to handle the larger volume of data 
generated. Continuing with the computational 
system for spectral file analysis (reported last 
year), further developments have been made. 
As many as 16 spectra can now be compared 
with the total file and those members which 
correspond at any one of three discrimination 
levels are obtained. Output by this procedure 
can be presented analyzed in various ways so 
as to identify groupings by clinical type and 
chromatographic characteristics and demon- 
strate any relationship within subgroups. Ap- 
plication of the computational system to analy- 
sis of chromatographic profiles has yielded 
helpful clarifications. Certain regions of the 
profiles are clearly common to the leukemia 
and controls; a few regions are present in the 
leukemia profiles which appear to have no 
counterpart in the controls; and certain regions 
are peculiar and do not correlate in a meaning- 
ful way with any others. 

Energy Metabolism Section 

Dr. Pratt and his associates, Drs. Morrison 
and Millar, have pursued their investigations 
of the patterns of heat production of rats with 
concomitant recording of feeding behavior and 
other activity. Their goal is to find the mode 
and extent to which feeding behavior (pat- 
terns) influences heat production and, in par- 
ticular, how the changes in feeding and gen- 
eral behavior pattern of the tumor-bearing rat 
influence its energy exchange. They are also in- 
vestigating the relative changes in water and 
material exchange in tissue compartments of 
the rat during imposed and induced changes in 
food intake and the effect on these of tumor 
induction and growth. They have found that 
the relative constancy of the size of the activ- 
ity compartment of total energy expenditure 



42 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



has been consistently confirmed in normal rats 
at normal temperatures. Energy expenditure of 
normal rats made aphagic by lesions in the 
lateral hypothalamus increases along with a 
disruption of normal patterns of differentiated 
activity. In rats in which the aphagia is sus- 
tained, the size of the activity compartment 
of total energy is greatly increased. 

Dr. Pratt, in collaboration with Mrs. Toal 
and Mr. William C. White, is continuing to de- 
velop mathematical and computer program- 
ming techniques for application in biomedical 
problems. Ultraviolet spectrophotometric anal- 
ysis of oligonucleotides has employed this tech- 
nique previously for the determination of the 
base composition and evaluation of sample 
purity of oligonucleotides. This has been ex- 
tended to determine nucleotide sequences. The 
'technique involves obtaining spectroscopic 
data before and after enzymatic hydrolysis of 
mono-, di-, tri- and tetra-nucleotides of known 
sequence from which precise difference spec- 
tra are computed for each compound, utilizing 
mathematical (linear) programming solution 
methods. The difference spectra of oligoribonu- 
cleotides containing two or more adjacent 
adenylic acids were found to have a charac- 
teristic pattern, the magnitude of which was 
linearly related to the number of such adenylic 
acids present. Difference spectra sufficed to 
identify base composition and sequence for 
oligoadenylic acids. 

Dr. Pratt has been in charge of the com- 
puter operations of the Laboratory. The IBM 
1620 Data Processing System with 40K digits 
of memory, 2 disk storage drives and a line 
printer have been operated with prime time 
being shared by Dr. Reid, Mr. White and Mrs. 
Toal, with occasional use by Dr. Draper and 
Mr. H. King. A major portion of the computer 
time available was occupied with information 
storage files and search and retrieval tech- 
niques. The organization of the file of NCI 
research grants was modified to achieve faster 
loading and more efficient retrieval and output 
procedures. About one-fourth of the active 
Cancer grants have been loaded and comple- 
tion of the file is awaiting the installation of 
an additional disk storage drive and the re- 
mainder of the punched paper tape input rec- 



ords. A storage and retrieval system for the 
abstracts of pharmacology papers presented 
at the 1965 FASEB meetings was organized 
and implemented. Programs were written to 
organize the file and supply output for several 
requests from Dr. Shannon. The information 
file of medical histories and clinical records 
from the chorio-carcinoma chemotherapy 
study has been initiated and the conversion 
and storage of data has been started. The or- 
ganization of the file is such that a search and 
retrieval on 121 lines of patient medical his- 
tory will be available and the identification 
and retrieval of specific clinical tests on any 
date for any patient from a list of more than 
165 different tests and spanning several years 
and many admissions can be made. 

Physical Biology Section 

Dr. Shack continues his studies on the char- 
acterization of the nucleic acids and nucleopro- 
teins of normal and malignant tissues in terms 
of their physical, chemical, metabolic and bio- 
logical properties. These studies entail the de- 
velopment of additional procedures for isola- 
tion, fractionation and characterization of 
nucleic acids and nucleoproteins. Specific ob- 
jectives within this framework include the de- 
termination of whether (a) DNA of malignant 
cells, including those of viral origin differs 
from that of normal cells, such as deletion of 
a normal component, addition of an abnormal 
component or a possible detectable change of a 
normal component, and (&) does a "metasta- 
ble" DNA serve as a template for DNA synthe- 
sis. He has improved methodology so that sub- 
microgram amounts of DNA can be resolved by 
electrophoretic methods. These new procedures 
include (a) carrier systems containing unla- 
beled native and denatured DNA or (b) trace 
amounts of labelled native and denatured DNA 
as aids in the characterization of various 
DNA's. Using such procedures, partially rena- 
tured DNA was electrophoretically resolved 
from both the native and denatured forms. The 
type of product formed during renaturation 
of DNA depends on the conditions of original 
denaturation (temperature, salt concentra- 
tion). Renaturation of DNA, denatured at 
high salt concentration, gives a product con- 



NATIONAL CANCER INSTITUTE 



43 



sisting of both denatured and partially rena- 
tured DNA, while denaturation at low salt con- 
centration leads to a single species which ap- 
pears to be a partially renatured aggregate. 
Using infected tissue cultures as a source ma- 
terial, it has proved possible by such proce- 
dures to separate renatured polyoma DNA 
from the tissue DNA which remains denatured. 
Dr. Breitman and research associate Dr. 
Cannon are studying control mechanisms of 
purine and pyrimidine ribo- and deoxyribo- 
nucleotides. In collaboration with Dr. S. 
Perry and research associate Dr. R. A. Cooper, 
studies on pyrimidine metabo^sm in human 
leukocytes were conducted. It was observed: 
(1) The percent DNA-thymine derived from 
exogenous deoxythymidine increased from 13% 
to 87% over a range of deoxythymidine con- 
centrations from 0.03/x M to 30(V M. (2) De- 
oxythymidine caused expansion of the total 
deoxythymidine di- and tri-phosphate pool but 
did not influence the contribution of the path- 
way de novo to this pool. Hence, the increas- 
ing contribution of exogenous deoxythymi- 
dine to the formation of DNA-thymidine oc- 
curred because of a progressive dilution of 
deoxythymidine triphesphate synthesized de 
novo with deoxythymidine triphosphate de- 
rived from exogenous deoxythymidine. (3) In 
concentrations above 0.3 /jM, deoxythymidine 
inhibited DNA synthesis but not RNA synthe- 
sis. This inhibition was dependent on the 
continued presence of deoxythymidine in the 
medium and was reversed by the addition of 
deoxycytidine. (4) The incorporation of deoxy- 
thymidine- 3 H into deoxythymidine di- and tri- 
phosphate pools of intact leukocytes from nor- 
mal donors and patients with chronic myelo- 
genous leukemia reached a maximum within 
5 minutes at 37° C. A steady state was main- 
tained during the subsequent 20 minutes at 
37° C. The time to reach maximum incorpora- 
tion of deoxythymidine- 3 H into the deoxythy- 
midine monophosphate pool was slower than 
into the deoxythymidine di- or tri-phosphate 
pools. In leukemic cells, the rate of equilibrium 
of deoxythymidine monophosphate with deox- 
ythymidine- 3 H was related directly to the de- 
oxythymidine concentration and inversely to 
the temperature. (5) With increasing concen- 



trations of deoxythymidine, expansion of the 
deoxythymidine monophosphate pool derived 
from deoxythymidine was greater than expan- 
sion of the deoxythymidine di- and tri-phos- 
phate pools. In leukemic leukocytes the deoxy- 
thymidine di- and tri-phosphate pools were 
approximately equal in size and accounted for 
65% of the thymine deoxynucleotides in the 
presence of 1 /*M of deoxythymidine but only 
10% in the presence of 300 pM deoxythymi- 
dine. In normal leukocytes at these deoxythy- 
midine concentrations, the values were 35% 
and 5% respectively of the total thymine de- 
oxynucleotides. (6) Over the range of deoxy- 
thymidine concentrations studied, incorpora- 
tion of deoxythymidine- 3 !! into thymine deoxy- 
nucleoside-monophosphate pool was 2-5 fold 
greater and incorporation into thymine deoxy- 
nucleoside di- and tri-phosphate pool was 6-15 
fold greater in chronic myelogenous leukemia 
than in normal leukocytes. Incorporation of 
3 fiM. of deoxythymidine- 3 H into DNA was 30- 
fold greater in chronic myelogenous leukemia 
than in normal leukocytes. The results indi- 
cate that human leukocytes have the enzymat- 
ic capability to convert thymine to deoxythy- 
midine. This enzymatic capability is not ex- 
pressed in the absence of added deoxynucleo- 
sides because of rapid catabolism of deoxythy- 
midine and thymine and a limited supply of an 
available deoxyribose source. 

Radiation Biology Section 

Dr. Elkind and Dr. Francesco Mauro (Guest 
Worker) are studying the mechanism of the 
repair of X-ray damage of cells grown in tis- 
sue culture. Changes in protein, DNA and 
RNA synthesis and the effects on these syn- 
theses of drugs which affect repair of X-ray 
damage are under investigation. They have 
developed a model for the fractionated dose 
response of cultured mammalian cells which 
they have named a Repair-Progression model. 
The repair part starts promptly after the first 
dose. This also occurs at lowered temperature 
and in the absence of oxygen. The progression 
part also starts promptly but is temperature 
sensitive and can be retarded at 37° C by 
either inhibitors of protein synthesis, a defi- 
cient medium, or the absence of oxygen. Re- 



44 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



suits obtained with asynchronous as well as 
synchronous cells (synchronized by exposure to 
hydroxyurea) support the general features of 
the model and, hence, a study of the fractiona- 
tion responses to be divided into the repair and 
progression parts can be made. In addition, 
the influence of drugs on changes in repair 
and/or progression can be studied. DNA 
synthesis apparently is not required for repair. 
Inhibition of DNA synthesis can be produced 
by high concentration of thymidine (1-3 mM). 
Under these conditions, division stops after G 2 
cells have divided, and from gross chemical 
measurements very little DNA is synthesized 
(less than 10%) in the ensuing 10 hours. The 
advantage of thymidine as a DNA inhibitor is 
that cell viability is not greatly affected. Using 
high concentrations of thymidine to inhibit 
DNA synthesis in synchronous and asynchro- 
nous cells, they observed that the "coupling" 
between DNA synthesis and X-ray response 
is stronger for first dose responses than for 
second dose responses (i.e., after sublethal 
damage repair). Thus it appears that even if 
X-ray response and DNA synthesis are tightly 
bound before a first dose, irradiation serves to 
uncouple this relationship. . 

Dr. Riesz has been continuing work on the 
effects of ionizing radiation on macromolecules 
of biological importance: In collaboration with 
Dr. F. H. White, Jr., of the Heart Institute, the 
distribution of free radicals in the gamma ra- 
diolysis of several dry proteins has been ex- 
amined. Gamma radiolysis of dry proteins in 
vacuo and subsequent exposure to tritiated hy- 
drogen sulfide, a radical interceptor, leads to 
the formation of carbon-tritium bonds. It has 
been shown that the observed tritium distribu- 
tion determined by amino acid analysis and 
scintillation counting, corresponds closely to 
the free radical distribution. Ribonuclease, car- 
boxymethylated reduced ribonuclease, lyso- 
zyme, chymotrypsinogen A, insulin and gelatin 
have been investigated. A comparison of all the 
proteins irradiated shows that in every in- 
stance methionine, proline and lysine have 
very high specific activities, while valine, iso- 
leucine and phenylalanine contain little trit- 
ium. However, each protein has, within this 
general pattern, its own characteristic radical 



distribution. The ratio of the highest to lowest 
specific activity for a given protein ranges 
from 20 for insulin to about 70 for ribonu- 
clease. Contrary to past interpretations of elec- 
tron spin resonance spectra, glycine radicals 
do not play a dominant role as free radical 
sites on carbon. Dr. Riesz has also studied the 
mechanism of the protective effect of certain 
metal ions in the y-radiolysis of dry metal- 
ribonuclease complexes. The reduction of cu- 
pric ions during the y-radiolysis of Cu 2+ -ribo- 
nuclease complexes was measured as a func- 
tion of dose and of Cu 2+ /ribonuclease ratio 
and compared with the survival of enzymatic 
activity. The results of experiments at rt^m 
and at liquid nitrogen temperatures support 
the proposal that metal ions protect because 
of their ability to act as interceptors of sub- 
excitation electrons and/or of hydrogen atoms. 

Dr. Smith is pursuing her studies on the 
kinetics of recovery of hemopoietic cells in 
irradiated animals. Employing the assay of 
the Till and McCulloch transplantation meth- 
od (counting colonies formed in the spleen), 
it has been possible to study the radio-protec- 
tive effects of such agents as /3-mercaptoethyl- 
amine, bacterial endotoxin, colchicine and 
other antimitotic agents. While colchicine 
seems to have a beneficial effect similar to that 
of endotoxin when given after irradiation, it 
has a moderate to pronounced effect on femoral 
colony forming units, marrow cellularity, per- 
ipheral leukocyte counts and survival. It ap- 
pears that the initial depressant effect is nec- 
essary for the subsequent early recovery ob- 
served when colchicine is given 1-3 days before 
irradiation. Studies on changes in peripheral 
blood and splenic colony formation using vin- 
blastine are being undertaken with tumors of 
low radiation sensitivity (lymphosarcoma 
1798) and the Bruce lumphoma. 

Miss Uphoff is undertaking studies in the 
field of immunogenetics of tissue transplanta- 
tion. She is developing new methods of alter- 
ing the immune response as well as studying 
the possibility of modifying tissue antigenic- 
ity. In addition, a study of cell to cell or cell- 
antigen interaction in the initiation of the im- 
mune response is under investigation. In these 
investigations radiation is used chiefly as the 



NATIONAL CANCER INSTITUTE 



45 



immunosuppressant agent. Transplantation of 
hematopoietic tissue into irradiated mice is 
used to test donor and host response, since 
mechanism, sensitivity and specificity of tissue 
reactions can be demonstrated by appropriate 
selection of various inbred strains or hybrid 
combinations. Tolerance is demonstrated either 
by skin or tumor homograft test systems. Stud- 
ies on genetic factors influencing irradiation 
protection by bone marrow have indicated that 
the early deaths reported in two different 
strain combinations following mid-lethal X-ir- 
radiation and homologous bone marrow inocu- 
lation, were found to be unique to these two 
strain combinations (CBA vs C57BL and 
BALB/c or C 57 BL). If the inoculation of 
homologous bone marrow is delayed or if one 
pre-immunizes the X-irradiated host, early 
death patterns are altered. She has demon- 
strated that a simple gene difference at the 
H-2 locus between donor and host strains was 
found to be sufficient to account for the severe 
"secondary disease" following lethal X-irradi- 
ation and bone marrow therapy. Differences at 
H-l provide variable reactions, while differ- 
ences at H-3 produced no secondary disease. 
Immunologic tolerance to tumor homographs 
could be induced by pretreatment with ameth- 
opterin and antigen (viable cells from appro- 
priate strain). She has also demonstrated a 
chimera consisting of three genotypes. 

Dr. Maxwell is studying the effect of ioniz- 
ing radiation upon the mammalian cell both 
in vivo and in vitro and determining the role 
of RNA and DNA in radiation damage. He has 
shown that in the early stages the surviving 
ability of an irradiated suspension of rat liver 
ribosomal particles to synthesize (in vitro) 
acid-insoluble protein is approximately a log 
function of the radiation dose. Doses of Co 60 
y-rays of the order of 150,000 rads reduce the 
synthesizing ability by a factor of two. This 
factor is approximately the same for both en- 
dogenous directed synthesis and artificial mes- 
senger (polyuridylic acid) directed synthesis. 
It also appears to be independent of the pres- 
ence or absence of dissolved oxygen in the 
irradiated solution. 

Dr. Draper has previously reported that nat- 
ural hemolysin production in rabbits is se- 



verely suppressed for about 10-14 days follow- 
ing a single exposure of 500 R whole-body 
irradiation. A subsequent recovery stage en- 
sues during which there is a renewal of natural 
antibody production at a relatively rapid rate. 
Enhanced serum titers are attained between 
the third and fourth postirradiation weeks. 
The recovery phase is usually accompan- 
ied by a change in the avidity of the antibody 
but the direction of the change is largely un- 
predictable. The production of highly avid 
natural hemolysin in some rabbits precludes 
the distinction between natural antibody and 
that induced specifically by immunization with 
sheep erythrocytes. In contrast to an active 
immune response, the production of natural 
hemolysin continues to be radiosensitive even 
during the rapid production stage during re- 
covery. This may be explained by the natural 
hemolysin titers representing the sum of a se- 
ries of short-term, weak, individual primary 
responses to the normally occurring hetero- 
phile antigens in small but constant supply 
from the rabbit's environment. This series of 
responses can be interrupted by moderate 
doses of X-rays. During the recovery stage it 
is likely that a larger population of cell is pro- 
ducing antibody. Such cells would be expected 
to be more radioresistant. Irradiation during 
the recovery phase often results in a delayed 
depression of titer indicating that the radio- 
sensitive cells are those exposed to antigen 
after irradiation. The spleen participates ac- 
tively in natural hemolysin formation. Studies 
are in progress concerning the production and 
nature of antibodies produced against differ- 
ent physical forms of the same antigen. 

Office of the Chief 

Dr. Blnm has pursued his studies in hyper- 
plasia induced in mouse epidermis by ultravio- 
let light. Methods of measuring hyperplasia 
are quite difficult. Illumination with visible 
light after irradiation with ultraviolet light 
increases the hyperplastic response. Hyperpla- 
sia occurs in the epidermis of the ventral, un- 
exposed surface of the mouse ear, concurrently 
with that, in the epidermis of the dorsal sur- 
face which is directly exposed to ultraviolet 
light. This suggests that the increase in cell 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



proliferation is mediated by a diffusible sub- 
stance, since the ultraviolet light does not pen- 
etrate sufficiently to account for this effect. 
Injections of homogenates prepared from nor- 
mal and irradiated epidermis cause epidermal 
hyperplasia when injected into a local vein of 
the ear. Homogenates from irradiated ears pro- 
duce the greater hyperplasia, indicating that 
some specific substance is released by the ir- 
radiation. 

Dr. White together with Dr. Millar have been 
studying the role of a growing tumor as a 
stressing agent in protein metabolism. When 
pregnant rats are inoculated with the Walker 
256 carcinosarcoma subcutaneously, effects ob- 
served are dependent on the time the tumor 
was inoculated. When female rats are inocu- 
lated with the tumor 7 days prior to evidence 
of pregnancy, the fetuses of all mothers are 
either resorbed or born dead. Rats with tumors 
less than 7 days of age when pregnant, deliver 
young which are normal in size. However, the 
inability of the mother to supply adequate milk 
results in animals which are stunted (about 
50%) at weaning. These animals remain stunt- 
ed for 3 weeks before they appear the same 
weight as rats born from non-tumor-bearing 
animals. It appears that the rat with a 3-week 
tumor (prior to parturition) cannot consume 
enough food to satisfy the growth of the tu- 
mor and supply protein for the formation of 
milk. In this competition, the tumor obtains 
what it needs at the expense of the lactating 
gland. Pregnant rats with 2- or 1-week tumors, 
deliver normal litters and the offspring are 
essentially normal at weaning. With Dr. Bates, 
a study on the nitrogen excretion and oxygen 
consumption of Fischer rats bearing a mam- 
matropic tumor has been made. Tumors im- 
planted intramuscularly in these rates grow 
slowly. After 40 days, this tumor begins grow- 
ing. A tremendous quantity of protein is ex- 
creted by these rats and there is an increase 
in oxygen consumption. Although the total ni- 
trogen increases, urea values remain constant. 
Food intake of the tumor-bearing rats in- 
creases 2-3 fold. Paired feeding experiments 
are in progress to determine the effect of in- 
creased protein intake on protein excretion. 



The machine, glass blowing, and electronics 
shops, headed by Mr. Mencken, Mr. LeBrun, 
and Mr. Coffey, respectively, continue to make 
many research problems practical. The ability 
of these men to study the problems of the Lab- 
oratory and to design operational equipment 
not on the market has resulted in inestimable 
progress. In addition, their staffs have done 
an endless amount of emergency repair and 
modification jobs which not only have saved 
valuable time but have prevented the failure of 
many experiments. These unsung heroes have 
been an important arm in the structure and 
progress of the Laboratory. 

DERMATOLOGY BRANCH 

The clinical and laboratory research pro- 
gram of the Dermatology Branch has contin- 
ued along lines developed in recent years with 
the addition of two new laboratory activities, 
namely research in cell biology and electron 
microscopy. The major groups of the research 
interests are: (1) Mycosis Fungoides Lym- 
phoma, (2) Epidermal Growth and Differen- 
tiation, (3) Biology of Lymphoreticular Cells, 
and (4) Melanogenesis. 

Mycosis Fungoides Lymphoma 

Immunologic reactivity of patients with this 
disease remains normal throughout the course 
of the lymphoma. Whereas patients with other 
lymphomas, particularly Hodgkin's disease, 
have been found to be immunologically hypo- 
responsive, patients with Hodgkin's disease 
show no immunologic impairment, particu- 
larly demonstrable in studies of skin sensiti-. 
zation to materials such as dinitrochloroben- 
zene (DNCB) and the ability to reject homo- 
grafts. Diseases other than lymphoma have 
been studied for comparative immunologic re- 
sponsivenesses. These diseases have included 
leprosy and Sjogren's disease, both of which 
show marked impairment in capacity to react 
allergically to DNCB. Patients with leproma- 
tous leprosy have been found to be more aner- 
gic than patients with non-lepromatous dis- 
ease. 

Interesting phenomena has been observed in 
patients with Mycosis Fungoides who have 



NATIONAL CANCER INSTITUTE 



47 



developed cutaneous allergic sensitivity to topi- 
cally applied nitrogen mustard HN 2 . When 
such patients are given 1 mg. of HN 2 intra- 
venously, cutaneous sensitivity to HN 2 disap- 
pears. This requires further exploration, par- 
ticularly in regard to other materials since 
such a desensitization might have useful clini- 
cal applications. 

Epidermal Growth and Reactivity 

The hypothesis that basal cell tumors result 
from basal cells' inability to keratinize re- 
mains valid and is further supported by addi- 
tional studies. These studies have tested the 
ability of basal cell tumor cells to participate 
in healing processes, a process requiring a cell 
to keratinize. There has been no evidence that 
basal cell tumor cells have capabilities of re- 
epithelializing wounded skin. A futher dem- 
onstration of the keratinizing defect in basal 
cell tumor cells has been found when podophyl- 
lin is applied to cutaneous tumors. Whereas 
normal epithelium manifests a high order of 
keratinization in response to podophyllin man- 
ifests a high order of keratinization in re- 
sponse to podophyllin, basal cell tumor cells 
show no such response. 

Attention is called to the successful tissue 
culture of human skin, an achievement which 
has been difficult or impossible in the past by 
many investigators. When split thickness spec- 
imens of skin are put into culture, epithelium 
grows quite readily and remains morphologi- 
cally normal over periods of several weeks. 

The response of basal cell tumors to topi- 
cally applied 5-fluorouracil (5-FU) has been 
confirmed. The selective response of lesions, 
compared to uninvolved skin, seems to be due 
to the ease of penetration of drug through the 
damaged epidermis overlying lesions. 

Melangonesis 

The ability of a soil micro-organism to pro- 
duce a dark pigment by utilizing coumarin as 
a substrate seems to be due to the ability of 
the organism to convert coumarin to tyrosine, 
thence tyrosine to melanin. The series of syn- 
thetic and degradative steps involved in the 
conversion of coumarin to tyrosine have been 



studied. Of particular interest is the conver- 
sion of O-coumaric acid to melilotic acid, which 
utilizes an oxidoreductase enzyme requiring 
FAD as co-factor. The enzyme melilotic hy- 
droxylase, converting melilotic acid to dihy- 
droxyphenyl propionic acid, has been purified 
to the extent that it migrates homogenously 
on gel-electrophoresis. It has a molecular 
weight of about 65,000. 

ENDOCRINOLOGY BRANCH 

In the fall of 1965, the Endocrinology 
Branch was reorganized with somewhat less 
than half of its previous staff, laboratory 
space, and clinical facilities. This has necessi- 
tated some reductions in program. Several 
areas of the previous research program are 
being continued under other auspices and this 
report will summarize those projects only that 
have remained within the National Cancer In- 
stitute. 

The appreciation of the complexity of andro- 
gen secretion and metabolism has led to the 
use of better mathematical models and systems 
for analysis. In conjunction with the develop- 
ment of new methods for the measurement of 
plasma androstenedione and dehydroepiandro- 
sterone, it has become possible to describe com- 
pletely the multiple origins and relative im- 
portance of each in the maintenance of plasma 
androgen levels. Such methods have been ap- 
plied to the study of diseases involving the 
gonads and to the analysis of the metabolic 
activity of several steroid-producing cancers. 

Further definition of the hormonal milieu, 
particularly with respect to such cancers as 
those of the breast and uterus, awaits the de- 
velopment of more sensitive methods for meas- 
uring plasma estrogens and their metabolites. 
We are attempting two entirely different ap- 
proaches to this important problem. 

Our group has maintained an interest in 
the endocrine effects and therapy of pituitary 
tumors. The direct assay of pituitary trophic 
hormones and better methods of assessing 
neuro-endocrine relationships have resulted in 
some new concepts of hypothalamic-pituitary 
controls. It has become clear that gonadotro- 
pin activity is not necessarily the first func- 
tion of the pituitary to be lost with increas- 



48 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



ing destruction of the gland. Further, it has 
been shown that these tumors may cause sig- 
nificant inhibition of pituitary function by in- 
terference with hypothalamic regulation 
rather than encroachment on the gland itself. 

Functional tumors of the endocrine glands 
have continued to interest our group. Pathways 
of biosynthesis in a luteoma were examined 
and the importance of the A 5 -pathway for 
synthesis and the role of the sulfoconjugates 
were delineated. The hypoglycemia produced by 
a functional adrenocortical cancer and the 
characteristics of a gonadotropin producing 
carcinoma of the bronchus were explored. 

Since endocrinology is concerned with the 
regulation of tissue growth and function, it 
is appropriate for the clinical endocrinologist 
to study regulatory mechanisms at the bio- 
chemical level. The initial studies have used a 
mammalian system, the chick oviduct, because 
a specific protein, avidin, is produced in re- 
sponse to a specific hormone, progesterone. 
Conditions for in vitro synthesis of avidin have 
been defined and methods for measuring small 
amounts of avidin have been devised. 

Our group has been strengthened this year 
by a visiting scientist from Australia. In the 
coming year, in addition to our normal com- 
plement of clinical associates, we shall have 
one third-year clinical Associate and a visiting 
scientist from Kyoto. Dr. Chrambach, a pro- 
tein chemist, has joined us recently and his 
skills should complement several ongoing pro- 
grams. We look forward to an expansion of the 
effort of the Branch during the coming year 
with increasing emphasis on the exploration of 
the hormonal milieu in relation to normal and 
abnormal tissue growth. 

IMMUNOLOGY BRANCH 

Two significant advances during the year 
profoundly influenced investigations in the Im- 
munology Branch. 

Development of the C'l Fixation and Trans- 
fer test by Drs. Borsos and Rapp in the Im- 
munochemistry Section has played a major 
role in antibody-cell antigen studies. The C'l 
Fixation and Transfer test has been applied 
in the Immunochemistry Section to theoretical 
and practical problems. It has made possible 



quantitative studies of cell antigens and of 
antibodies reacting with cell antigens, and has 
facilitated investigation of antibody: cell an- 
tigen reactions. One 18S IgM antibody mole- 
cule was shown to be sufficient to fix comple- 
ment and initiate cell lysis, but two 7S IgG 
antibody molecules must react with relatively 
closely spaced antigens to achieve the same 
effect. IgM and IgG, but not IgA, antibodies 
were shown to fix complement when reacting 
with human red cells. Differences in human 
Al and A2 erythrocyte antigens have been 
demonstrated and investigations of human 
leukocyte ( ?transplantation) antigens have 
begun. Viral tumor specific antigens were de- 
tected in studies of murine tumors induced by 
viruses. Applications of the C'l fixation and 
transfer technic should prove very valuable 
in studies of transplantation antigens, "tumor" 
antigens and genetic (and environmental) 
regulation of cell membrane composition. 

Immunoglobulin (gamma globulin) forma- 
tion in vitro in continuous culture of estab- 
lished human cell lines was demonstrated for 
the first time. IgG (7S immunoglobulin) and 
IgM (18S macroglobulin) formation has been 
accomplished. These cultures will be useful for 
basic biologic and biochemical study, but they 
may be most useful if antibody activity can 
be achieved. Specific human antibody synthe- 
sis in continuously cultured cells is now a goal 
of the Immunology Branch. 

The research activities of the Immunology 
Branch extended beyond the areas noted above. 
29 publications for the four Senior Investi- 
gators are listed in the Annual Project Reports. 
Studies of cell, especially tumor, antigens have 
been extended. A program of transplantation 
studies was initiated. The mechanism of com- 
plement activation and cell damage by immune 
processes continue to be investigated. 

Immunodiagnostic facilities for multiple 
myeloma, macroglobulinemia and immunoglo- 
bulin deficiency syndromes were established 
through a contract with Melpar. Currently 
about 40 serums are tested each week. Studies 
of immunoglobulin structure and function have 
been extended. 

The Immunology Branch provides forums for 
continuous education and exchange of infor- 



NATIONAL CANCER INSTITUTE 



49 



mation for NCI investigators through two reg- 
ular weekly seminars — one on Molecular Im- 
munology, and one on Cellular Immunology. 
Also, tutorial seminars in Immunology and 
Immunochemistry were conducted by NCI Im- 
munology Branch staff in both Autumn and 
Spring terms. 

METABOLISM BRANCH 
Amino Acid Transport 

In an attempt to characterize further amino 
acid transport mechanisms in fetal rat cal- 
varia, the effect of puromycin dihydrochloride, 
an inhibitor of protein synthesis at the ribo- 
somal level, was studied. Inhibition of protein 
synthesis, measured as glycine incorporation 
into T.C.A. precipitable protein was approx- 
imately 90% at 10 ug/ml of puromycin hy- 
drochloride. The transport of alpha-amino 
isobutyric acid (AIB) was inhibited over a 
range of 10-600 ug/ml following a two-hour 
incubation in puromycin dihydrochloride. 
Ouabain and sodium cyanide have been shown 
previously to inhibit AIB uptake in fetal rat 
calvaria. If this inhibition is studied after 
varying incubation times in inhibitor followed 
by a standard 30 minute labeled amino acid 
uptake, there is initial inhibition of 35% for 
cyanide and 15% for ouabain when inhibitor 
and amino acid are added together. The rate 
of inhibition versus time slows on prolonged 
incubation. In contrast, when puromycin is 
used as inhibitor, there is small if any initial 
inhibition and the percent inhibition versus 
time forms a straight line on semi-log paper. 
A half-life for the transport process can be 
graphically determined at about 120 minutes. 
Active transport of lysine, a dibasic amino 
acid, was not inhibited by any concentration 
of puromycin dihydrochloride (10 ug/ml-600 
ug/ml) and no inhibition could be seen over 
a 180-minute incubation period. Lysine uptake 
could be inhibited by cyanide in a manner 
analogous to that seen for AIB uptake. These 
findings suggest that puromycin can inhibit 
active transport of amino acids by inhibiting 
the synthesis of necessary proteins, and differ- 
ences in the catabolism of these proteins allow 



for further characterization of transport sys- 
tems. 

Investigations have been carried out evalu- 
ating the relationship between amino acid 
transport and collagen synthesis. A steady 
state model has been utilized where fetal rat 
calvaria are incubated in 0.14mM Proline for 
three hours and then carrier free Proline-C 14 
is added for varying time intervals. It has 
been shown that AIB, hydroxy-proline and 
L-azetidine-2-carboxylic acid decrease the in- 
tracellular free amino acid proline pool, protein 
synthesis and collagen synthesis. In addition, 
lowering the proline concentration below 
0.14mM results in decreased collagen synthe- 
sis. Dipeptides such as glycylproline, glycylhy- 
droxy-proline, hydroxyprolylglycine, and pro- 
lylglycine do not inhibit amino acid uptake. 
This suggests that although hormones may 
regulate the general level of cellular metab- 
olism, a secondary control of protein synthesis 
by the extracellular amino acid milieu is of 
importance. At present, efforts are underway 
to construct a multicompartmental mathemati- 
cal model for collagen synthesis so that quan- 
titation of effects of varying substances at 
specific steps in protein synthesis can be car- 
ried out. 

Calcium Metabolism 

A multicompartmental analysis for calcium 
kinetics has been developed. Metabolism bal- 
ance data combined with Ca 47 disappearance 
from blood, cumulative radioactivity in urine 
and feces, and surface counts from two sites 
are processed on an IBM 70904. By a least 
squares method of curve fitting, pool sizes and 
turnover rates may be calculated. Parameters 
which may be interpreted physiologically are 
bony accretion, bony resorption, urine and 
fecal turnover rates and gastrointestinal ab- 
sorption. 

During the past year, application of this pro- 
gram has concentrated on the mechanisms of 
calcium homeostasis. A series of 8 hypopara- 
thyroid patients were studied. Four of these 
patients were studied twice; once as a control, 
the second time at a new steady state pro- 
duced by injections of purified parathyroid 
hormone. In the hypoparathyroid patients, 



50 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



there was found a decrease in miscible cal- 
cium pool and a decrease in resorption of bone, 
but bony accretion, urine and fecal turnover 
rates were not significantly different from nor- 
mals. After being perturbed by parathyroid 
hormone, there were increases in bony resorp- 
tion as well as in urine and fecal turnovers. 

Other studies included three normal volun- 
teers on low and high calcium intakes. While 
the gastrointestinal tract was adequate in con- 
trolling the amount absorbed from high Ca + + 
intake, a low (200 mgm/day) calcium diet 
produced a negative calcium balance with in- 
creased bony resorption. The urinary and en- 
dogenous fecal turnovers were only slightly 
decreased. No significant change was seen in 
bony accretion. 

A patient with calcinosis universalis was 
also studied and found to have a large miscible 
calcium pool with slow exchange rates between 
compartments. The urinary and fecal turn- 
overs were also small. Bony accretion and re- 
sorption were not significantly different from 
normal. These changes were correlated with his- 
tochemical and crystallographic studies. 

The existing computer analysis is for a 
steady state system; a new computer pro- 
gram for analyzing the nonsteady state is being 
prepared. With this method, hormone depen- 
dent parameters for calcium control may be 
calculated. The feedback relationship between 
serum ionized calcium and parathyroid hor- 
mone production may be investigated in man. 

Porphyrin Metabolism 

8-aminolevulinic acid synthetase (ALA 
synthetase) has been partially purified from 
mitochondria. Heme and other metallo-porphy- 
rins have been shown to produce inhibition of 
this enzyme in vitro only at concentrations 
above 5xlO _4 M, casting some doubt on the 
significance of heme as a functional inhibitor 
in vivo. 

The kinetics of induction of hepatic ALA 
synthetase have been studied alone with the 
effects of inhibitors of protein synthesis (pur- 
omycin, actinomycin D, 5-fluorouracil, p-fiuoro- 
phenylalanine, etc.). Glucose blocks induction 
of this enzyme as effectively as actinomycin D. 
A mathematical model of enzyme synthesis 



has been developed which allows the calcula- 
tion of the half life of the specific messenger 
RNA for a given enzyme. Using this model 
the half life of ALA synthetase is 67-74' and 
that of its messenger RNA is approximately 
the same, i.e., 40-70'. 

It has been shown that heme, the end prod- 
uct of the pathway which ALA synthetase 
controls, will prevent induction of ALA syn- 
thetase when injected intravenously at certain 
times before induction is initiated. This is the 
first direct evidence of heme as a repressor 
of ALA synthetase in mammalian tissues and 
supplements the indirect evidence from the 
tryptophane pyrrolase work reported last year. 

When heme is injected intravenously, the 
level of hepatic ALA synthetase oscillates 
markedly for periods of 4 to 5 days after a 
single dose. The oscillations can be greatly 
amplified by administration of inducer 5 hours 
before sacrifice. This probably is + he first dem- 
onstrated example of significant oscillations of 
the level of an enzyme. The only other exam- 
ple is reduced nicotinamide adenine dinucleo- 
tide oscillations in in vitro yeast extracts of 
glycolyzing systems. The oscillations of ALA 
synthetase in mammalian liver explain Wat- 
son's observations on variations in bilirubin 
excretion following heme administration and 
raise the question of the role of ALA syn- 
thetase in biologic clock mechanisms. 

The fact that ALA dehydrase (the enzyme 
following ALA synthetase in the porphyrin 
pathway) also oscillates after heme adminis- 
tration is evidence of a genetic operon con- 
trolling the two enzymes. A mathematical 
analysis of the kinetic factors controlling the 
amplitude and periodicity of the oscillations 
of these enzymes is in progress. 

The elevation of serum protein bound iodine 
in acute porphyria has been found to result 
from elevation of the serum level of thyroid 
binding globulin. 

Glucose loading tests in patients with acute 
porphyria have shown frequent abnormalities 
in the form of a decreased rate of glucose 
disappearance from the blood and abnormally 
increased blood pyruvate levels. 

A paradoxical response of blood levels of 
growth hormone has been demonstrated in 



NATIONAL CANCER INSTITUTE 



51 



seven patients with acute porphyria after glu- 
cose loading. The level of growth hormone 
rises rather than decreasing as seen in nor- 
mals. The syndrome of inappropriate ADH in 
acute porphyria has been demonstrated by di- 
rect assay of blood and urine for the hormone. 
These growth hormone and ADH abnormali- 
ties occur in most patients and indicate the 
frequency of abnormal hypothalamic physiol- 
ogy in acute porphyria. 

The effects of diet on preventing the chem- 
ical abnormalities of acute porphyria pre- 
viously demonstrated in the experimental ani- 
mal have been extended to patients. A de- 
creased caloric intake can initiate attacks of 
the disease and high carbohydrate diets can 
end these attacks. 

A small fraction of women whose attacks of 
acute porphyria recurred cyclically with their 
menstrual periods have been maintained free 
of disease by use of oral anovulatory hormone 
preparations. 

Nucleic Acid 

To estimate the amount of virus DNA in 
cells, radioactive RNA complementary to this 
DNA was prepared in vitro. This RNA was 
then reacted with DNA prepared from cells 
infected with or transformed by DNA viruses 
and the amount of DNA-RNA hybrid forma- 
tion was measured. DNA preparation from 
hamster cell lines transformed by SV40 virus 
had an increased amount of DNA complemen- 
tary to SV40 RNA, if and only if "T" anti- 
gen was demonstrable by immunofluorescence. 
This is evidence for correlation between "T" 
antigen and persistence of viral genome. 
AGMK cells infected with either adeno-7 virus 
alone, or adeno-7 and SV40 virus produced 
rather similar amounts of virus DNA, al- 
though the titer of infection virus was 100- 
1000 higher in the infected cells. Thus, the 
"enhancement" by SV40 occurs at a level be- 
yond DNA synthesis. 

The same DNA-RNA hybridization-tech- 
niques have been applied to the taxonomy of 
mycoplasma and streptococci. By this tech- 
nique, the mycoplasma are a very heterogene- 
ous group of organisms. No relatedness was 
evident between M. pneumoniae and the other 



mycoplasmas tested. Other human mycoplasma 
fall into a number of species with only a low 
level of interspecific relatedness. Within some 
species, and of M. hominis I, there appeared 
to be much greater heterogeneity between dif- 
ferent isolates. Other groups such as M. pneu- 
moniae or the "FS" group were not detectably 
heterogeneous. Similar studies of the strepto- 
cocci showed excellent correlation between nu- 
cleic acid homology and serologic typing. Dif- 
ferent subgroups of streptococci were related 
and serotypes within a species were distin- 
guishable. 

Studies of adeno infected KB cells have dem- 
onstrated the appearance of an RNA com- 
ponent (VA-RNA) localized in the high speed 
supernate of the cell cytoplasma but distinct 
from transfer RNA. This has been partially 
characterized with regard to molecular prop- 
erties, metabolic properties, and base sequence 
in oligonucleotide maps. Somewhat similar 
RNA components are extractable from ribo- 
somes of uninfected cells. Preliminary studies 
by oligonucleotide analysis and nucleic acid 
hybridization techniques indicate these are not 
identical to VA-RNA. 

Metabolism of Plasma Proteins 

The known heterogeneity in the chemical 
nature of the antibody globulin molecules is 
paralleled by a markedly heterogeneous pat- 
tern of distribution synthesis and survival. In 
subjects without diseases affecting immuno- 
globulins, the survival half time of IgD was 
2.8 days, of IgA 6.1 days, of IgM 5.1 days, and 
of IgG 24 days. Approximately 45 percent of 
the IgG and IgA molecules are distributed 
within the intravascular pool, while 70 to 80 
percent of IgD and IgM molecules are within 
this pool. The catabolic pathway of each of 
these four major classes of immunoglobulins 
was shown to be completely independent of the 
other proteins and determined by the F c seg- 
ments of the immunoglobulin molecules, the 
part that does not contain the antibody-com- 
bining site. 

The immunoglobulin levels were studied in 
the sera of 70 patients with thymomas. Nine 
such patients were shown to have marked re- 
duction of IgG and IgA, with frequent recur- 



52 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



rent infections. Seven of these nine subjects 
had a marked reduction in IgM level. The two 
remaining subjects had extreme elevations of 
IgM comparable to that seen in the macro- 
globulinemia of Waldenstrom. These disorders 
of IgG, IgM and IgA concentration were shown 
to be predominantly disorders of immunoglo- 
bulin synthesis. In all but one of the patients 
with a reduced IgG concentration the IgG sur- 
vival was considerably prolonged. In the one 
remaining subject gastrointestinal protein loss 
was superimposed on the hypogammaglobu- 
linemia. One patient with Sjogren's syndrome 
and lymphoma was shown to have a pre- 
viously undescribed pathophysiological mecha- 
nism causing extreme reduction in the serum 
IgG concentration. This patient had a complex 
cryoglobulinemia that required the simultane- 
ous presence both in IgM and IgG molecules. 
The patient's own 19 S macroglobulin was re- 
quired for cryoprecipitation. The coprecipitant 
IgG required could be from any human source. 
Three factors contributed to the low estimate 
of the IgG levels. First, cryoprecipitation of 
serum IgG during quantitation of the levels 
in vitro at room temperature resulted in spur- 
iously low estimates for the serum concentra- 
tion. However, even when these studies were 
done under conditions that prevent cryoprecip- 
itation, the patient still had an exceedingly 
low serum concentration of IgG. This was 
shown to be due to a combination of defec- 
tive synthesis of IgG, and accelerated cata- 
bolism of IgG. The patient had a normal sur- 
vival of albumin and normal IgM, and thus 
had an isolated hypercatabolism of one pro- 
tein, IgG, presumably secondary to its forma- 
tion of aggregates with the cryomacroglobulin 
in vivo. 

Studies on the serum protein metabolism of 
patients with myotonia dystrophica were con- 
tinued. Patients with this disorder were shown 
to have a reduced concentration and total body 
pool size of IgG secondary to a unique heredi- 
tary disorder of the immunoglobulin catabo- 
lism and isolated hypercatabolism of IgG with 
normal IgG synthesis. Both the patients' and 
normal IgG were catabolized at the accelerated 
rate in the patients while the patients' IgG 
was catabolized at a normal rate in normal 



subjects. All other proteins studied, including 
ceruloplasmin, albumin, IgA, IgD and IgM, had 
a normal survival in these subjects. 

Studies on the protein metabolism of pa- 
tients with ataxia telangiectasia were con- 
tinued. These patients usually had a marked 
reduction or absence of serum IgA, but had 
a protein similar to IgA in the saliva. It was 
shown that the reduction in IgA seen in these 
patients was secondary to an extreme reduc- 
tion synthesis of this protein, and in some 
cases, hypercatabolism of this protein. It was 
shown that there is normally little or no trans- 
port of IgA from the serum into the salivary 
secretion, but that the very high levels of IgA 
normally seen in excretory secretions is prob- 
ably derived from specialized plasma cells in 
the mucosal and submucosal surfaces of the 
respiratory and gastrointestinal tract. The IgA 
is then coupled with a transfer piece in glan- 
dular structures and secreted as a first line of 
defense against infections entering the body. 
It should be noted that the patients with 
ataxia telangiectasia have a very high inci- 
dence of reticuloendothelial neoplasms. 

Subjects with the Wiscott-Aldrich Syn- 
drome, a disorder characterized by eczema, 
thrombocytopenia, a very high incidence of 
infections, and sex-linked recessive inheri- 
tance, have a markedly reduced level of IgM, 
a normal or increased level of IgG, and a mark- 
edly elevated serum concentration of IgA. 

Patients with the syndrome of intestinal 
lymphangiectasia were shown to have a short 
survival of all proteins studied, including IgG, 
IgA, IgM, ceruloplasmin albumin and fibrino- 
gen secondary to loss of these proteins into the 
gastrointestinal tract. The synthetic rate for 
each of the immunoglobulins was normal and 
the patients were able to produce antibodies 
to Vi and tularemia antigen. Marked lympho- 
cytopenia was noted in these patients pre- 
sumably due to loss of lymphocytes into the 
bowel secondary to the disorders of lymphatic 
channels. The patients with lymphopenia 
showed marked skin anergy to skin test anti- 
gen. Skin grafts from unrelated donors are sur- 
viving for over one year and second set grafts 
from the same donor are also intact at two 
months. 






NATIONAL CANCER INSTITUTE 



53 



A pattern of low immunoglobulins and skin 
anergy was also demonstrated in subjects with 
other causes of gastrointestinal protein loss 
associated with disorders of intestinal lymphat- 
ics, including Whipple's disease, constrictive 
pericarditis, and regional enteritis. Subjects 
with extreme gastrointestinal protein loss, 
without disorders of gastrointestinal lymphat- 
ics, such as subjects with sprue, allergic gas- 
troenteropathy, vascular diseases of the bowel, 
and ulcerative colitis, did not have lymphocy- 
topenia or anergy. Gastrointestinal protein 
loss was demonstrated for the first time in 
subjects with chronic vascular disease of the 
bowel, with the blind loop syndrome and in a 
patient with generalized myopathy and con- 
gestive heart failure. 

Studies on the role of the kidney in the 
metabolism of fractions of the immunoglobu- 
lins were extended. It was shown that the kid- 
ney plays a major role in the catabolism of 
human Bence-Jones proteins of either the 
lambda or kappa type, of normal human L 
chains, and of the L chain type of Bence- 
Jones protein formed secondary to multiple 
myeloma in mice. A number of other proteins 
that appear in the urine do not share this 
catabolic pathway. Specifically, the F ab and F c 
fragments of 7S gamma globulins, the Bence- 
Jones proteins of mice made up of both H 
chain and L chain, and those that are frag- 
ments of the H chain alone were not catabol- 
ized by uptake into the proximal convoluted 
tubule of the kidney. It is suggested that the 
H chain of immunoglobulin molecule protects 
the L chain from renal tubular metabolism. 

Previously it has been shown that the frac- 
tion of the circulating IgG catabolized per day 
is dependent on the concentration of this pro- 
tein, in contrast to the situation observed with 
IgA, IgM, ceruloplasmin and fibrinogen where 
the serum concentration of the protein does 
not affect the fractional catabolic rate. With 
IgG in man the fraction of the intravascular 
pool catabolized ranges from 2 percent in pa- 
tients with agammaglobulinemia to 16 percent 
in patients with multiple myeloma and a high 
level of IgG. Similar hypercatabolism of IgG 
has been demonstrated in mice following the 
infusion of IgG from a number of species or 



the F c fragments of the gamma globulin mole- 
cule. Similarly, the F c fragments survival is 
affected by the concentration of the intact 
IgG molecule. It was shown that enzyme induc- 
tion is not required for this hypercatabolism 
in response to infusion of the protein, since 
this phenomenon continues when the animals 
are given high doses of Actinomycin-D or 
cyclohexamide, materials that inhibit messen- 
ger RNA and protein synthesis, respectively. 
Mathematical analysis of these studies indi- 
cates that the best explanation for this phe- 
nomenon is the presence of a saturable protec- 
tion system for IgG molecules. In contrast to 
the formulation of Brambell these studies in- 
dicate that the absolute amount of IgG pro- 
tected, increases with increasing concentration 
of IgG, while the fraction of the total num- 
ber of molecules protected decreases sharply. 
The reciprocal of the quantity of molecules 
protected plotted against the reciprocal of the 
total circulating pool size gives a straight line 
function. From this graph, the total number 
of protection sites and the affinity of the mole- 
cule for the protection sites can be defined. 
It is postulated that the same protection sys- 
tem specific for IgG responsible for the con- 
centration effect is also responsible for the 
facilitated transport of IgG across the new- 
born gastrointestinal mucosa in animals and 
across the placenta in man. 

Erythropoiesis 

The studies of the relationship between 
metabolic rate and erythropoiesis in the hy- 
pothyroid dog have been completed. These 
studies show that in the dog made hypothyroid 
with large doses of iodine that there is a nor- 
mochromic normocytic anemia characterized 
by a decreased rate of synthesis of red cells 
that this anemia could be repaired with thy- 
roxin but not with dinitrophenol. It could not 
be repaired with iron, copper, cobalt, folic acid 
or vitamin B 12 . Dinitrophenol raised the meta- 
bolic rate to normal or even greater than nor- 
mal levels in both normal and hypothyroid 
dogs but neither in the hypothyroid dog nor 
in the normal dog did this result in a sig- 
nificant effect in erythropoiesis. This is then a 
demonstration of the uncoupling of erythro- 



54 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



poiesis and metabolic rate. In addition, the 
hypothyroid dog develops a marked lipidemia. 
This is corrected on the administration of 
thyroxine and dinitrophenol. Thus the altera- 
tions in blood lipids that accompany the hypo- 
thyroid state can be repaired by elevation of 
the metabolic rate to the normal by both thy- 
roxine and dinitrophenol. 

The initial stages in the development of a 
multicompartment (nine-pool compartment) 
model of iron metabolism utilizing the com- 
puter facilities of the Office of Mathematical 
Research of NIAMD have been completed. It 
is anticipated that this will yield better in- 
formation regarding the rate of formation of 
red cells than the simple mathematical models 
currently being used. 

Studies of hemoglobin synthesis by reticu- 
locytes from thalassemic patients demonstrated 
that overproduction of alpha chains occurs in 
thalassemia minor as well as thalassemia 
major. In contrast, there was slight or no de- 
tectable overproduction of alpha chains in 
hemoglobin Lepore trait. There was a recip- 
rocal relationship between the specific activity 
rates of alpha to beta chain and that of alpha 
to delta chains, suggesting that, as the chain 
production was depressed, there was a stimu- 
lation of delta chain production. 

Bilirubin Metabolism 

The bilirubin clearance method has been de- 
veloped and sufficient data has been acquired. 
The data shows that the rate of disappearance 
of bilirubin from the plasma in the sixteen 
patients studied can be described in terms of 
two rate components; the first having a half- 
time in the range of 20-30 minutes and the 
second a range from 100-200 minutes. In gen- 
eral, the rapid component is associated with 
the bulk of the removal. A three compartment 
model has been developed to quantitate the 
bilirubin production. This has yielded values 
ranging from 139 to 1530 mg per day. The 
principal abnormality seen in these two rate 
components has been in two patients with Gil- 
bert's disease where the second component 
was somewhat longer but, more particularly, 
it was a more significant fraction of the total 



turnover. With this model it is now thought 
possible to measure bilirubin production. 

Other studies have shown that in two pa- 
tients with a thoracic duct fistula that there 
was a small transfer to the order of one-half 
a percent of the bilirubin from plasma through 
hepatic lymph to the thoracic duct lymph. This 
did not appear to be a significant pathway 
anatomically for bilirubin. 

In the turkey, an animal with a nucleated 
red cell, there is a non-red cell source of bile 
pigment that is significant. The data have not 
yet been fully analyzed for its quantitative sig- 
nificance. 

Other studies of bilirubin physiology have 
been concerned with the development of an 
assay for the measurement of hepatic glu- 
curonyl transferase using bilirubin in physi- 
ological quantities as a substrate. The male 
rat shows higher values than the female rat. 
The assay has been also carried out in six 
patients with clinical evidence of inability to 
conjugate bilirubin and the amount of enzyme 
has shown to be decreased. The significance 
of this method lies in the hypothesis that bili- 
rubin glucuronyl transferase may be a different 
enzyme than other glucuronyl transferases. In 
the past measurements have used other sub- 
strate materials to determine the amount of 
glucuronyl transferase. The data may not be 
directly extrapolatable to the situation with 
bilirubin if another substrate is used. 

A series of biochemical studies have been 
undertaken to explain the mechanism of bili- 
rubin toxicity. The principal accomplishments 
have been a demonstration that unconjugated 
free bilirubin uncouples oxidative phosphory- 
lation. When unconjugated bilirubin is com- 
plexed with albumin this biochemical lesion is 
repaired in the liver and kidney but not in 
the brain. This is a possible explanation for 
the development of neurological lesions in 
kernicterus. 

The metabolism of bilirubin was studied in 
the kernicteric Gunn rat. This rat has an in- 
herited lack of hepatic glucuronyl transferase. 
This is manifest by a marked hyperbilirubi- 
nemia and in some animals by the neurological 
signs of kernicterus. On the kernicteric animal, 
bilirubin-C 14 could be demonstrated in the 



NATIONAL CANCER INSTITUTE 



55 



brain after intraperitoneal administration, 
while in equally jaundiced, but not neurologic- 
ally involved animals, bilirubin-C 11 could not 
be found in the brain. This animal and bili- 
rubin-C 14 made it possible to study the prob- 
lems of bilirubin and kernicterus. 

SURGERY BRANCH 

The Surgery Branch of the National Cancer 
Institute continues to act in a dual capacity 
carrying out its own specific clinical and lab- 
oratory investigations and providing consulta- 
tive surgical service to other branches of the 
National Cancer Institute and the clinical units 
of each of the other institutes of the National 
Institutes of Health. Because of the broad scope 
of investigative clinical studies being carried 
out by the various institutes, this service has 
included urological, Otolaryngol ogical, and 
gynecological as well as thoracic and general 
surgical consultations. 

During the previous twelve months, 1,240 
surgical consultation requests were received 
and answered by the Surgery Branch Staff. 
Four hundred and twenty-nine of these were 
from the National Cancer Institute, while 811 
were from clinical units of other institutes. 
These numbers were original requests and do 
not take into account the minimal five-fold 
increase that would exist if the repeated visits 
to each patient were recorded. Three hundred 
and eleven operative procedures were per- 
formed as a result of these consultations, 112 
were from the medical units of the Cancer Insti- 
tute and 199 were performed on other institute 
patients. Forty-two percent of these procedures 
were major surgical endeavors. Consultants 
from the community saw 286 patients, 268 of 
whom were non-malignant otolaryngeal prob- 
lems. These consultants performed or assisted 
in only 12 surgical procedures. 

A total of 705 surgical operations were per- 
formed by the staff; 345 were major proce- 
dures and 360 were minor procedures. Surgery 
Branch admissions resulted in 394 surgical 
procedures, of which 63% were major opera- 
tions. 

The clinical interests of the Surgery Branch 
continue to be varied, and this is in part dic- 
tated by the type of patient referred. Since 



disease which can be controlled by standard 
surgical procedures is being handled in the 
community, 85% of the admissions were pa- 
tients with advanced but regionally localized 
disease not suitable for the usual surgical 
therapy. Sixty-eight percent of the admissions 
had received prior radiation, surgery or chemo- 
therapy and were treatment failures. 

Malignant disease of the head and neck con- 
tinues to be a prime interest. In past years 
head and neck cancer has constituted approx- 
imately one third of the Surgery Branch pa- 
tient care load. However, it has been gradually 
increasing and during this past year has ac- 
counted for 52% of the patient admissions. 
This increase might be in part due to the in- 
creased awareness of the dentist to malignant 
oral-pharyngeal disease and the apparent in- 
creased prevalence in heavy smokers, con- 
sumers of alcohol and "snuff dippers". During 
the past year, 15 patients have been direct 
referrals from the National Institute of Dental 
Research. The study of the effectiveness of pre- 
operative radiotherapy for oral, pharyngeal 
and laryngeal cancer continues. Radiotherapy 
is given 24 hours preoperatively on a random 
basis according to the double blind technique. 
The number of patients is low because of the 
limitation requiring previously untreated pa- 
tients. The 1000 roentgens given preopera- 
tively is based upon several of our laboratory 
studies which showed that metastases could 
be decreased significantly by this treatment 
in animal tumor systems by this therapy. 
Clinical experience to date indicates that 1000 
roentgens in a single dose preoperatively is 
the maximum dosage which can be used and 
not have significant wound morbidity. 

Survival by conventional means of therapy 
from paranasal sinus cancer is veiy poor be- 
cause of frequent tumor extension into the 
ethmoid, sphenoid and pterygoid areas, result- 
ing in local recurrence in spite of the usual 
surgical or radiological techniques of treat- 
ment. Thirty-one patients have had a combined 
intracranial transfacial en bloc resection of 
the paranasal sinus area. Resection includes 
the cribriform plate and medial orbital walls 
and usually leaves the orbital contents intact. 
Operative mortality has been 7%. Follow-up 



56 



ANNUAL RFVIEW OF INTRAMURAL RESEARCH 



for from 4 months to 9 years indicates that 
19 patients have remained disease free. 

The morbidity often associated with exten- 
sive head and neck surgery has become less of 
a problem. The National Institute of Dental 
Research continues to furnish these patients 
with meticulous day by day refitting - of well 
constructed prosthetic appliances. Through 
their cooperative efforts, we have been able to 
avoid the prolonged and repeated hospitaliza- 
tions so often associated with plastic recon- 
struction. In so doing, the operative defect may 
be carefully followed for local recurrence 
rather than have it hidden by an overlying 
skin pedicle. Morbidity has been further de- 
creased by the judicious use of antibiotics, 
cervical esophagostomy feeding intubation and 
better placed skin incisions. Even though ra- 
diation recurrent lesions constitute 71% of the 
head and neck material and surgery is in no 
sense less radical, there has not been a carotid 
hemorrhage in 28 months. 

We have been exploring the feasibility of 
full dose preoperative radiotherapy followed 6 
weeks later with definitive surgery. Patients 
thus far chosen for this pilot venture have pre- 
sented large localized, malignant lesions and 
have been estimated to have an expected 5- 
year survival of less than 3 to 5%. 

Referred patients with carcinoma of the cer- 
vix continue to have had previous definitive 
surgery, supracervical hysterectomy, irradia- 
tion and histories of never having had a Papa- 
nicolaou smear. Our patients with advanced 
disease treated with extensive surgery show an 
operative mortality of less than 10% and an 
overall five-year survival of 43%. The patients 
who underwent radical hysterectomy have a 
70% survival, anterior pelvic exenteration, 
34% and total pelvic exenteration, 24%. With 
the development of better techniques for pa- 
tient selection the survival rate following 
radical surgery continues to improve. Unilat- 
eral leg edema, sciatic distribution of pain, 
bilateral renal involvement, lymphangiog- 
raphy, renal and surgical physiological studies 
are all clinical and diagnostic aids useful in 
determining feasibility of surgery. For se- 
lected advanced tumors of the pelvic organs, 
vagina, cervix, uterus bladder and rectum, 



surgery continues to offer the best chance of 
cure, but this is not done without, at times, 
considerable morbidity. Improved techniques of 
blood volume determination, red cell survival 
studies, bowel sterilization isotope renography 
and vital sign monitoring have provided an in- 
creased understanding of postoperative physi- 
ology and have permitted more knowledgeable 
postoperative care. All have combined to make 
radical surgery a practical means of bringing 
advanced pelvic disease under control. 

Experience with over 500 distal extremity 
lymphangiograms, indicate that this technique 
of demonstrating lymph nodes is an interesting 
research tool, but is of little real diagnostic sig- 
nificance in the surgical patient. Its value as 
seen by this surgical group is first, to draw 
the surgeon's attention to an area suspicious 
of lymph node involvement with tumor, and 
second to allow a more complete lymph node 
dissection for the surgeon infrequently in- 
volved with radical lymph node resection. 
The false positive and false negative interpre- 
tations greatly outnumber the incidences of ac- 
curate interpretations. In many patients this 
may be due to altered lymphatic drainage sec- 
ondary to previous radiotherapy. 

The ileal conduit continues to be the most 
satisfactory means of urinary diversion fol- 
lowing total pelvic exenteration. This technique 
results in few complications and preserves ex- 
cellent renal function in nearly all instances. 
However, attention must be directed toward 
continual antibacterial prophylaxis in order to 
prevent infectious complications. Of interest 
has been 13 patients who have undergone uri- 
nary diversion in spite of preoperative and op- 
erative evidence of unilateral ureteral occlu- 
sion. High transection and urinary diversion 
into an ileal conduit has resulted in all but 
one of the kidneys regaining function. In half 
the patients very satisfactory function re- 
turned, in the others improvement was noted 
but a variable degree of impairment remained. 

Radical cancer surgery is by its very nature 
extensive surgery and carries a relatively high 
mortality and morbidity. However, our experi- 
ence with over 100 radical hysterectomies, 50 
anterior exenterations, 250 total pelvic exen- 
terations and 4 combined amputative proce- 



NATIONAL CANCER INSTITUTE 



57 



dures (2 of which are alive over 2 years) sug- 
gest that within the proper environment, this 
procedure should be offered to more patients 
with advanced pelvic disease. With intensive 
colostomy and urinary ileostomy instruction 
and guidance and vaginal reconstruction in se- 
lected instances, these patients who have had 
radical pelvic surgery can be restored to es- 
sentially a normal, pleasant and productive life. 

An increasing number of extremity sar- 
comas, most of which are recurrent following 
previous treatment are being seen by the Sur- 
gery Branch. Selected instances of amputation 
and particularly hemipelvectomy which allows 
removal of all extremity muscular and ten- 
donous insertions and origins have been per- 
formed with no mortality, a high degree of 
physical rehabilitation and an increased 3- 
year survival free of disease. 

A small, but significant number of patients 
are referred each year with malignant mela- 
noma. We are reviewing the perfusion studies 
and the surgical approach practiced in other 
centers. Our practice of wide local excision and 
lymph node dissection continues to be the treat- 
ment of choice with selected patients being 
referred for perfusion and surgery. When we 
have in, 41 patients, a 5-year survival of 16 
patients and a 10-year survival of only 6 pa- 
tients, there is indication for incorporating 
well protocolled studies into the regime of this 
disease. With this consideration in mind, the 
Surgery Branch has developed a chemotherapy 
protocol in animals with S-91 melanoma. The 
limiting effects of methotrexate therapy are 
bone marrow depression and gastro-intestinal 
toxicity. Primate studies have been conducted 
to determine whether small amounts of citro- 
vorum factor given by hypogastric arterial in- 
fusion will selectively protect the pelvic bone 
marrow against the effects of intravenously 
administered methotrexate and yet not gen- 
erally circulated to interfere with the sys- 
temic anti-tumor effects. In addition, attempts 
to modify the gastro-intestinal tract toxicity 
by administration of non-abosrbabe intestinal 
antibiotics have been undertaken. At this time 
it appears that both of these premises are valid 
and that with their implementation much 



larger doses of methotrexate can be safely 
given. 

A long-term survey of infectious complica- 
tions following cancer surgery shows that the 
patient who carries a pathogenic organism with 
him into the operating room, whether it be 
from the skin, nose, or throat or parcularly 
within the tumor, runs a significantly in- 
creased risk of developing an infectious com- 
plication postoperatively. Staphylococcia in- 
fections predominate, but are more easily con- 
trolled and cause less patient morbidity than 
other organisms. Antibiotic therapy remains 
routine with all patients undergoing cancer 
surgery, chloramphenicol being the most sat- 
isfactory and frequently used drug. This drug, 
has been extensively used for 6 years by the 
Surgery Branch without evidence of hematolog- 
ical complications. Using high dosage for a 
limited ten day interval which includes the 
preoperative, operative, and postoperative pe- 
riods, infectious complications have decreased 
during the past year to 7% as compared to 10- 
15% without preoperative coverage and 22- 
30% without any antibiotic therapy. During 
1965 staphylococcal infections accounted for 
only 36% of our infections and there were no 
deaths due to this organism. Staphylococcal 
infection certainly increases morbidity, but it 
is the pseudomonas, E. coli, Candida albicans 
and proteus organisms which have consist- 
ently been associated with the most resistant 
infections and the septic deaths. 

During the past year a fruitful cooperative 
clinical study has been carried on in associa- 
tion with the Experimental Therapeutics 
Branch, NHI. Eight patients with pheochro- 
mocytoma have undergone surgery following 
biochemical evaluation. From the care of this 
group of patients has emerged a program for 
relatively simple yet quite effective manage- 
ment of a clinical problem previously consid- 
ered formidable. 

Serial measurement of red cell volume, plas- 
ma volume and extracellular fluid volume has 
demonstrated that (1) following extensive sur- 
gery there is no evidence for a slowly circu- 
lating or non-circulating portion of the red 
cell volume; (2) post-operatively after exten- 
sive surgery there is a disproportionate plas- 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ma volume deficit present which has heretofore 
been unrecognized and is as yet unexplained; 
(3) post-operatively there is a deficit in the 
extravascular extracellular fluid volume sug- 
gesting the need for additional amounts of 
fluid at surgery. 

This laboratory has not been satisfied with 
the usual bio-assay methods of detecting anti- 
diuretic hormone and in the past 18 months 
has developed an immuno-assay technique ca- 
pable of measuring one hundred micromicro- 
grams of anti-diuretic hormone per ml. of solu- 
tion. Continued improvement in the sensitivity 
and precision of the technique is anticipated 
and then comprehensive physiological studies 
will be undertaken. 

During the past year, little attention has 
been directed toward further refinements in 
techniques of isolating tumor cells from the 
blood of cancer patients. It remains apparent 
that if enough blood is sampled from the can- 
cer-bearing patient, tumor cells will eventually 
be isolated. Surprisingly enough little correla- 
tion has been noted between patient survival 
and the presence or absence of circulating 
tumor cells. Tumor cell isolation and identifica- 
tion from wound washings and drainages has 
similarly been unrewarding in most respects. 
Possibly due to better techniques of filtering 
and certainly due to better criteria of identifi- 
cation the incidence of tumor cells has de- 
creased to such a low percentage as compared 
to previous years that validity of the overall 
study is questioned. A review of wound drain- 
age studies and survival in 170 patients shows 
no correlation as to the instance of local re- 
currence. This study has been unrewarding and 
suggests that an approach to local tumor re- 
currence through topical agents may not be 
successful no matter what agent is used. 

The cooperative, prospective study of pri- 
mary therapy of endometrial carcinoma should 
provide an answer to an old problem. The 
different types of therapy under study are sur- 
gery alone, preoperative radiation and surgery, 
and surgery and postoperative radiation. The 
long delay in starting this project continues, 
but patients will be assigned to therapy at 
random. Utilizing the Surgery Branch, as the 
Study Center, data from cooperating institu- 



tions will be collected and analyzed. The study 
is planned to include 400 patients. Ultimate 
analysis of the data after the completion of 
the five year follow-up will provide meaning- 
ful information concerning the relative effec- 
tiveness of the methods of therapy under 
study. 

A technique for transplanting endocrine 
glands in isologous animal systems has been 
used to study not only a state of endocrine 
hyperfunction but also the ability to regulate 
the animals endocrine activities through hor- 
mones produced by these organs. With these 
methods a physiological model has been used 
to study the inter-relationships between the 
parathyroid and thyroid glands in calcium 
homeostasis. Using hypercalcemic challenged, 
inbred rats and combining thyro-parathyroi- 
dectomy and parathyroid gland transplanta- 
tion at a remote site, further evidence has 
accumulated on the physiological role of a cal- 
cium lowering hormone. Preliminary evidence 
of a thyrocalcitonin-releasing factor from 
the parathyroid gland has also been demon- 
strated. In a similar experiment the function 
of the pineal gland and its relation to the men- 
strual cycle in normal females was shown. Ad- 
ditional studies using sympathetic cervical 
ganglia have demonstrated the histologic and 
biochemical survival of these tissues. A model 
was thus found which facilitates the study of 
the effects of pharmacological agents such as 
reserpine and tyramine on the release of nor- 
epinephrine. Observations on the effect of the 
VX 2 carcinoma in rabbits and on its ability to 
produce hypercalcemia are being made in an 
attempt to determine if the calcium imbalance 
is due to an excess of parathyroid hormone 
which may be produced by this tumor. 

Experiments are being carried out to evalu- 
ate the comparative abilities of oncogenic DNA 
viruses to induce neoplastic transformation in 
newborn hamster endocrine tissues in vitro. 
Transformation of pituitary, pineal thyroid, 
parathyroid, adrenal, testicle, and ovary tissue 
in explant cultures to malignant cells by Sim- 
ina virus 40 (SV40) and LEE 46 strain of 
adenovirus. These tumors are being evaluated 
for evidence of endocrine function and studies 
are being conducted for either a bio-assay or 



NATIONAL CANCER INSTITUTE 



59 



direct chemical assay of the tumors for specific 
hormones. 

A biological system for studying - tumor en- 
hancement with a specific fraction of the iso- 
logous immuno-globulins has been established. 
This consists of producing a potent antiserum 
in DBA strain of mice by repeated inoculations 
by EL4 ascites tumor from C57/BL mice. 
The antiserum is then fractionated by column 
chromatography and electrophoresis to see 
which of the specific immuno-globulins is re- 
sponsible for the enhancement phenomenon of 
these tumors across the H2 histocompatibility 
line. 

Further studies have been carried out in 
solid tumor patients subjected to thoracic 
duct lymph drainage to ascertain whether cel- 
lular or humoral factors in human lymph are 
responsible for suppression of the immune re- 
sponse. This has been done by taking the lymph 
from patients and subjecting it to sterile cen- 
trifugation to separate the lymphocytes from 
the protein fractions of lymph and then the 
protein fractions have been returned to the 
patients. The immune response was studied in 
these patients by small skin homografts and 
their ability to respond to a primary antigen. 
It has been shown that under these conditions 
the immune response is dependent more on the 
amount of protein and the depletion of immu- 
noglobins than it is the loss of small lympho- 
cytes in humans. 

Through collaborative studies with the 
Army Missile Command and Eastman Kodak 
the role of laser energy as an oncolytic tool 
continues to be intensively investigated. Pulsed 
energy from both ruby and neodymium sources 
and continuous wavelength studies from both 
Argon and C0 2 sources show tumoricidal ac- 
tivity in rodent, dog and primate liver. The use 
of the continuous wavelength laser as a "light 
knife" has been investigated. We are pessimis- 
tic about its ultimate clinical use either with 
skin or soft tissue. It is true that relatively 
bloodless hemihepatectomines can be safely 
performed with the CW laser and a massive 
amount of hepatic tissue may be destroyed 
with the pulsed laser without significant toxic 
effects to the host, but practicability is doubt- 
ful at the present stage of laser "refinement." 



At 1000 joule, semi-portable laser is being used 
daily in our laboratory and a 4 module, 1000 
joule, neodymium unit will soon be installed. 
It is fair to say that we continue to look upon 
laser energy as the next innovation in cancer 
cure with "suppressed enthusiasm". 

Further studies in renal physiology have 
been performed which seek causes of renal vas- 
cular hypertension and better means of diag- 
nosis and surgical management. Further stud- 
ies of urinary oxygen tension (Up0 2 ) in dogs 
have been carried out before and had inversely 
related Up0 2 to medullary sodium reabsorp- 
tion under profound osmotic diuresis. Reduc- 
tion of glomerular filtration rate by clamping 
the reneal artery has a variable effect on Up0 2 
suggesting the medullary blood flow (MBF) 
has a significant effect on Up0 2 under these 
conditions. Under the conditions of these stud- 
ies, however, medullary sodium transport ap- 
pears to be the determinant of Up0 2 . Until an 
accurate method of measuring MBF is devel- 
oped, this variable will remain uncontrolled 
and unknown. 

An attempt to validate 133 Xenon renal clear- 
ances as indicator of medullary blood flow is in 
progress. The medullary component of renal 
blood flow has been derived from clearance 
curves in a consistent manner and agree sub- 
stantially with data reported by others using 
autoradiographic techniques. Further study is 
indicated before accuracy can be determined. 

To test the hypothesis that angiotensin is 
inactivated by the liver, splenorenal venous 
shunt was performed in dogs made hyperten- 
sive by clamping the renal artery. None of the 
animals sustained relief of hypertension after 
shunting. Clinical trial of this procedure in 
hypertensive patients is not warranted. 

Epinephrine infusion into the renal artery 
to create temporaiy ischemia has been per- 
formed in one patient and several dogs. Anoxic 
tissue is known to be less sensitive to radia- 
tion effects. Marked deminution of renal blood 
flow during infusion of minute amounts of 
epinephrine occurred. We hope to show that 
there is protection against radiation damage to 
the body and thus allow larger doses of radio- 
therapy to be given to abdominal neoplasms. 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Clinical studies in renovascular hypertension 
have included differential studies of kidney- 
function and Up0 2 radioisotope renography 
and phonocatheter recording of renal artery 
pulse. Aortogram and differential function re- 
main the most reliable methods of diagnosing 
surgically curable renovascular disease. 

The value of phonocatheter and Up0 2 stud- 
ies await larger clinical experience. The isotope 
renogram is useful in following ureteral ob- 
struction and in management of patients fol- 
lowing renal artery surgery, but is too nonspe- 
cific to be the sole screening test for renal ar- 
tery stenosis. The renogram has also been help- 
ful in managing cancer patients after pelvic 
exenteration procedures requiring ileal con- 
duit diversion. It is a more accurate test of 
renal function than is the excretory urograms 
and can be used in patients allergic to uro- 
graphic contrast medium. 

Collaborative studies of patients with cysti- 
nuria have demonstrated the value of d-peni- 
cillamine in preventing recurrence of cystine 
calculi. Studies of the mechanism of increased 
cystein excretion by kidney biopsy and in vitro 
amino acid transport and renal arterial-venous 
amino acid differences suggest that increased 
turnover of cystine, lysine and arginine within 
renal parenchyma may be the basic defect in 
this disease. 

Urinary lactic dehydrogenase activity was 
studied in hamsters during induction of renal 
adenocarcinomas. In none of the experimental 
or control animals has total LDH excretion 
risen above upper limits of normal, even when 
both kidneys were almost entirely replaced 
by tumor. This study confirms previous reports 
from this department that urinary LDH is not 
a useful screening test for urinary tract neo- 
plasms. 

The effect of gonadotropins, testosterone 
and estradiol on surgically unilateral crypt- 
orchid rats has been studied. Gonadotropins 
and testosterone in moderately large dosage for 
3 weeks did not appear to have had deleterious 
effects as judged by bilateral testis biopsy six 
weeks after the cryptorchid testis was returned 
to the scrotum. 

Intestinal perfusion may still be a method 
that permits repeated dialysis of uremic sub- 



jects over a long period. Emphasis is being 
placed on definition of the variables of the pro- 
cedure, including serum and perfusate electro- 
lyte concentrations, serum and perfusate osmo- 
lality, rates of intestinal exchange and transfer 
of electrolytes and other crystalloids and such 
factors as the flow rate and temperature of 
the perfusate solution. 

Demonstration of the physiologic events in 
surgically de-vascularized and re-vascularized 
ureters is in progress. Omental sleeves may pre- 
vent the high incidence of uretero-vaginal fis- 
tulae in women subjected to radical histerec- 
tomy for carcinoma of the cervix. 

The diagnostic significance of tetracycline 
fluorescence in early neoplasms accesible to 
ultraviolet endoscopy is controversial. It may 
be that tetracycline fluoresces only when che- 
lated to calcium in tissue fluid or within ma- 
crophages. This problem is currently being 
studied in New Zealand white rabbits with 
VX 2 carcinomas. The state of calcium metabo- 
lism is being varied with parathormone and 
with parathyroidectomy. 

Production of experimental and transitional 
cell carcinomas of the bladder with aniline 
dye products is well established. The similarity 
of these papillary carcinomas to tumors of vi- 
ral etiology is striking, yet, no experimental 
studies are on record to assess the role of viral 
"carcinogenesis" in papillary carcinoma of the 
bladder. Such a study is being conducted on 
neonatal hamster bladders and in tissue cul- 
ture employing polyoma, adenovirus 7 and 12, 
SV-40 and E46-M viruses. 

Serum leucine aminopeptidase values are be- 
ing studied in patients with normal pregnancy, 
multiple gestations and patients with tropho- 
blastic neoplasms. This is a follow-up of earlier 
work which suggested that the rise of values 
of this enzyme which takes place in normal 
pregnancy may be higher and/or earlier in twin 
pregnancies. Since it does not rise in patients 
with hydatidiform mole, further studies on its 
usefulness in making this differential diagno- 
sis are in order. The report that the increment 
in leucine aminopeptidase activity in the serum 
of women during pregnancy is sensitive to in- 
hibition with methionine is to be evaluated. 



NATIONAL CANCER INSTITUTE 



61 



A study of the immunologic relationship be- 
tween women with pregnancy or a trophoblas- 
tic tumor and their husbands cellular antigens 
has been carried out using the technique of 
lymphocyte transformation in mixed leuko- 
cyte cultures. Preliminary results show that 
lymphocytes from normally pregnant women 
do not transform when grown with their hus- 
band's leukocytes in as high a percentage as 
they do when grown with cells from an unre- 
lated male. This effect is not found in women 
with trophoblastic tumors. Further studies are 
being carried out to evaluate serial changes in 
each trimester of pregnancy and the changes 
that take place during and after chemother- 
apy of the trophoblastic neoplasms. The latter 
are being correlated with the response of the 
patients to chemotherapy. 

In order to evaluate the possibility of dam- 
age to the ovum in a resting Graafian follicle 
by tritiated thymidine given to study bone 
marrow cellular kinetics, radioautographs of 
the ovaries of previously treated Rhesus mon- 
keys were made and evaluated for evidence of 
tritium concentration in the ova or closely ad- 
jacent granulosa cells. These studies are in 
their initial phase. 

Immune suppression and its effect on homo- 
graft rejection and the growth pattern of 
transplanted human choriocarcinoma are be- 
ing studied in hamsters, mice and monkeys. 
Techniques used in the hamster to obtain this 
effect are the administration of drugs (Metho- 
trexate, Methotrexate and Citrovorum Factor, 
and Crotisone), a total body irradiation plus 
Cortisone and anti-thymocyte serum. In mice 
the effects of thymectomy alone and thymec- 
tomy followed by a course of anti-lymphocyte 
sera are being evaluated. Studies outlined in 
monkeys call for evaluating the effects of thy- 
mectomy alone, thymectomy plus anti-lympho- 
cyte sera treatment and also anti-thymocyte 
sera treatment alone. 

The evaluation and improvement of diag- 
nostic and operative culdoscopy has continued. 
The comparison of culdoscopy and X-ray gyne 
cography has resulted in the realization that 
either technique can provide satisfactory in- 
formation depending upon the availability of 
the techniques and the ability of the operators. 



The development of the intraperineal probe 
has converted the previously static, diagnostic 
culdoscopic examination to a dynamic one per- 
mitting the operator to palpate and manipu- 
late the visualized structures. The development 
of a simple and easy technique for positioning 
makes the procedure significantly shorter and 
easier on both the patient and the operating 
room personnel. The technique of ovarian bi- 
opsy performed under culdoscopic examination 
has been developed and perfected and provides 
a safe technique for obtaining ovarian tissue 
with entirely acceptable morbidity. 

The increased incidence of cystic ovarian 
enlargement and multiple pregnancy associ- 
ated with ovulation induction, using human 
menopausal gonadotropin (HMG or Pergonal) 
and human chorionic gonadotropin (HCG) 
suggest overdosage. To test this impression and 
to select the minimal ovulatory dose gradually 
increasing amount of HMG has been given to 
selected anovulatory or oligo-ovulatory patients 
and the dose response relationship noted. Re- 
sults indicate a sigmoid dose response relation- 
ship with a greater response being associated 
with larger doses. Minimal ovulatory doses 
have ranged from 450 to 1725 international 
units. Grouping of patients by diagnosis has 
resulted in a marked grouping of the minimal 
ovulatory doses. It would appear that continua- 
tion of the study would supply information 
which will permit Pergonal dosage to be se- 
lected for the individual patient with a mark- 
edly lessened chance of unwanted side effects. 

Ancillary studies which are underway as a 
part of the general Pergonal study include the 
measurement of human chorion gonadotropin 
in patients with Pergonal induced pregnancies, 
the early detection of plasma chorionic gona- 
dotropin in the first two weeks of gestation 
prior to the first missed menstrual period, de- 
termination of anti-Pergonal antibody in pa- 
tients treated with Pergonal and the determi- 
nation of the effects of Pergonal on plasma 
testosterone and androstenedione levels. 

The effects of pharmocologic doses of estro- 
gen on the reticuloendothelial system clearance 
rate of 131 I tagged aggregated human serum 
albumin and on the febrile response to endo- 
toxin and etiochloanolone is being studied in 



62 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



an attempt to elucidate the effects of this hor- 
mone on these physiolgic parameters. 

Ethiodol is a commonly used lymphangio- 
graphic radio-opaque medium. Preliminary 
carcinogenesis studies in mice suggested that 
this agent might have activity which would 
discourage its further use. These animal stud- 
ies made use of Ethiodol which was injected 
into three different sites. Subsequent animal 
investigations compared Ethicdol with the pop- 
py seed oil component of Ethiodol. The com- 
pleted data on these two oils shows that tu- 
mors appeared in mice at irregular intervals 
in all three injection sites. The response sug- 
gests that there may be some carcinogenic ac- 
tivity at the dosage level administered. Data on 
the pathological findings is now being assem- 
bled for statistical review. 

In the study of tumor metastases an accu- 
rate and reproducible method of identifying 
small tumor foci is necessary. Frequently the 
color of normal animal lung parenchyma and 
small experimental tumor implants is similar 
and it is difficult, if not impossible, to differen- 
tiate grossly between the two. Microscopic 
identification of tumor implants is often not 
practical because serial sections are expensive 
and the review is time-consuming. The accu- 
rate counting and sizing of small, lightly pig- 
mented tumor metastases in the lungs of ex- 
perimental mice have been facilitated by the 
development of a method whereby the lungs 
were insufflated with a 15% solution of India 
ink. After being washed with Feketes solution, 
which is a bleach preservative, these tiny im- 
plants, not detected in the unstained fresh lung, 
became discretely visible. The accuracy and 
time-saving features of this method aid in the 
verification of lung metastases and permit a 
much wider range of spontaneous and induced 
animal tumors in studies of experimental tu- 
mor metastases. 

A comparison study of survival, tumor 
growth, and metastases with transplanted, in- 
duced and spontaneous tumors in mice has 
shown that metastases observed in the three 
separate tumor systems were similar, in both 
amputated and intact tumor-bearing mice even 
though the growth rates of the tumor systems 
varied markedly. The similarity in the number 



of metastases observed is believed to be due to 
survival which was obtained by individual 
animal housing. On the basis of this study, it 
appears that the three tumor systems evalu- 
ated might be interchangeably used in metas- 
tases studies if recognition is given to their 
varied growth rates. 

The long term program on tumor metastases 
is in a transition period of orientation to the 
immunological approach to the understanding 
of local recurrence and metastases. Studies 
have been performed to investigate the nature 
of the immunity imparted by certain chemi- 
cally-induced tumors upon their hosts. Particu- 
lar emphasis has been focused on whether 
chemically induced tumor immunity exists in 
the autochthonous host, and whether the spe- 
cific immunity that we see in later transplant 
generations is due to antigenic alteration of 
the tumor in serial passages. The cellular lo- 
calization of this antigen seems to be in the 
nuclear fraction of the tumor tissue. With 
weakly antigenic tumors, i.e., S-91 melanoma, 
prior immunication with cell-free tumor ex- 
tracts had no significant effect on tumor 
growth. Enhancement occurred when this ex- 
tract was given at the time of tumor inocu- 
lation. 

Previously used methods for the study of 
tumor specific immunity have been extended 
to two new and unique tumor systems. A chem- 
cally-induced gastric adenocarcinoma has 
been found to be immunogenic, demonstrating 
an immunity which lasts at best, ten months, 
can be adoptively transferred by isologous 
lymphoid cells may be abrogated by splenec- 
tomy or large doses of tumor cells, and re- 
quires viable lymphoid cells for its effective- 
ness. Attempts to utilize immune lymphoid 
cells to alter the growth of established tumor 
have been only partially successful in that 
growth has been slowed but complete regres- 
sion has not been achieved. 

Similar immunologic phenomena have been 
demonstrated in another unusual system, a 
spontaneous fibrosarcoma. Immunotherapeutic 
experiments with this tumor of recent origin 
are in progress. 

Efforts to establish a number of new lines 
of visceral tumors in inbred rat strains are 



NATIONAL CANCER INSTITUTE 



63 



reaching fruition. Freely transplantable yet 
isologous tumors of visceral organs have not 
been studied in inbred rats, whose size is more 
appropriate to the evaluation of experimental 
operative surgery models than is the mouse. 

CHEMOTHERAPY 

The recent reorganization of the Chemother- 
apy Program has had a markedly unifying 
effect. The program plan (1), developed in last 
year's review of 1955-1966 experience, was 
approved by the National Institutes of Health 
and the National Advisory Cancer Council, and 
in September 1965 the new organization took 
form. 

The research strategy of the chemotherapy 
program is built around two objectives — the 
use of drugs to achieve selective killing of tu- 
mor cells and the search for new anti-tumor 
agents. 

Research on selective toxicity of drugs for 
the tumor cell advanced sharply during the 
past year. They key to this advance has been 
the development by Skipper of an animal model 
in which it is possible to estimate the number 
of tumor cells killed by drug and the number 
surviving. Drugs can be studied in the model 
to discover the optimal schedule for maximum 
tumor cell kill ("schedule sensitivity"). Bruce 
and his colleagues have studied the mitotic cy- 
cles of normal and of tumor cells in mice, and 
discovered differences that permit drug admin- 
istration to kill large numbers of tumor cells 
with slight effect on normal cells ("cycle sen- 
sitivity"). Merkle and colleagues have devel- 
oped a mathematical model that uses cycle and 
schedule sensitivity data in predicting curative 
drug regimens for a variety of human neo- 
plasms. The application of these new concepts 
to patients depends on ability to measure gen- 
eration times of tumor cells. Preliminary anal- 
ysis of those malignant diseases where drugs 
seemingly are curative, suggests that the bio- 
logical and mathematical models are predic- 
tive. The strategic objective of highest prior- 
ity at present is the study of selective toxicity 
of drugs in patients. 

Two correlative approaches support this ob- 
jective. One of these is a broad expansion of 



pharmacological studies of the known anti- 
tumor drugs. It is essential to know both the 
concentration of active drug at the target site 
and the time required at target for maximum 
tumor cell kill and minimal host toxicity. These 
studies are underway in both the Skipper 
model and in patients. Secondly, selective tox- 
icity can be enhanced by measures that pro- 
tect the host against the toxic effects of drugs. 
The most effective of these are platelet replace- 
ment and protection against the infections 
arising from drug induced host depletion. Re- 
search and development in these areas and in 
national resources have been sharply expanded. 
The second major objective is the discovery 
of new chemicals with anti-tumor activity. The 
method of search, now in its eleventh year at 
NCI has been thoroughly reviewed and sub- 
stantial changes invoked. There are over 30 
drugs (counting analogues) with substantial 
effects in clinical cancers. These are active in 
100 or more animal tumors. Examination of the 
screening data of 10 years shows that all but 
2 of these drugs are selected by two animal 
tumors, L1210 leukemia in the mouse and 
Walker 256 carcinosarcoma implanted intra- 
muscularly in the rat. The conditions of these 
two animal tests can be adjusted to that few 
if any false positives will be selected. The pri- 
mary screen for the search has been rede- 
signed and at present is under a dry run with 
all known clinically active and a number of in- 
active drugs. The new screen permits the drop- 
ping of a number of less satisfactory animal 
tumors and will provide both drug schedule in- 
formation and dose response data. The amounts 
of drug required are about one-fifth those 
needed in the old screen so that much of tissue 
culture screening (used for the frequently in- 
adequate amounts of sample) can be dropped. 
While the new primary screen ensures that it 
can pick up signals of anti-tumor activity of a 
wide variety of synthetic and natural products, 
exploration must go forward on screens of 
other types. Until clinical activity is shown for 
drugs that are inactive in the primary screen, 
this question cannot be settled. Drugs highly 
active in other animal tumors but inactive in the 
primary screen are being sought for introduc- 
tion into clinical trial. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The extent of the search for new agents has 
been under active debate. As long as the search 
is empirical, it would seem wise to allow con- 
siderable latitude. Since there are now a num- 
ber of effective synthetic agents, there is an 
opportunity to begin a study of structure activ- 
ity relationships. From this study it may be 
possible to concentrate synthesis efforts in the 
most rewarding areas. There have already been 
sharp reductions in studies of alkylating agents 
and anti-purines. At the same time, if the dif- 
ficulties of synthesis can be overcome, more 
needs to be done with the anti-folics and nu- 
cleosides. In the natural product areas there 
are a number of materials mostly of high mo- 
lecular weight highly active in the screen. The 
large size of these agents poses difficult prob- 
lems of purification and none of these materi- 
als has reached clinical trial. Since the known 
clinically active drugs are of relatively small 
molecular weight, perhaps these large mole- 
cules from plants may open a new era in can- 
cer chemotherapy. 

The new research on selective toxicity sug- 
gests the working hypothesis that large inter- 
mittent pulses of therapy are more damaging 
to tumor cells than to normal cells. If one ex- 
amines the best clinical results obtained with 
drugs, it is noted that these have in large part 
occurred with pulsed regimens. Remissions ap- 
proaching cure have been seen in choriocarci- 
noma, acute lymphocytic leukemia, Burkitt's 
lymphoma, childhood solid tumors and testicu- 
lar tumors. The drugs responsible include 2 
anti-metabolies (methotrexate and 6 mercapto- 
purine), an alkylating agent (cyclophospha- 
mide), a bacterial fermentation product 
(actinomycin D), 2 plant products (vinca alka- 
loids), and an animal hormone (corticoster- 
oids). Cure is a word to be used with care in 
connection with disease, since in its strictest 
sense, it means that a large sample of treated 
patients subsequently die off at the same rate, 
appropriately corrected for age and sex, as the 
general population. While too little time has 
elapsed since the recognition of selective tox- 
icity of drugs for tumors, to make a judgment 
about cure, 5 year complete remissions are suf- 
ficiently common to generate a strong suspi- 
cion about the ultimate conclusion. A number 



of other tumors are quite sensitive to these 
and other drugs. The present tactical problem 
is to apply the concepts of selective toxicity 
in each instance. 

In addition the search for new agents gives 
evidence of new weapons. Streptonigrin, mi- 
thramycin, daunomycin, dibromomannitol and 
cytosine arabinoside have recently been found 
clinically active. All of these are strongly pos- 
itive in the new screen, as are an increasing 
number of natural products not yet in produc- 
tion for clinical trial. 

Acute Leukemia Service 

The intensive chemotherapeutic regimen, 
POMP, has been completed with an excellent 
complete remission rate (90%) and a low 
treatment mortality rate (3%). The median 
remission duration thus far is 16 months. Mor- 
bidity was kept at a moderate level by the vig- 
orous use of platelet transfusions and antibi- 
otics. Between 500 to 1000 platelet and leuko- 
cyte transfusions are given monthly. There is 
very little question that the use of platelet tran- 
fusions has been accompanied by a dramatic 
reduction in the incidence of hemorrhage in 
thrombocytopenic patients. On the other hand, 
the effectiveness of leukocyte transfusions 
(largely cells from donors with chronic myelo- 
cytic leukemia) is not as clear. However, their 
efficacy is for the first time being evaluated in 
a controlled study. The answer should be avail- 
able within the next year. 

The importance of this very high remission 
rate in acute leukemia is that it strongly indi- 
cates a shift in emphasis in the treatment of 
this disease. A major effort now needs to be 
directed toward the eradication of the leukemia 
leukocytes remaining in the patient in remis- 
sion. Accordingly more recent protocol studies 
have been designed with this thought in mind. 

Solid Tumor Service 

The major disease emphasis in the Solid 
Tumor Service continues to be the lymphomas 
and chronic myelocytic leukemia (CML). 
Based on the successful use of combination 
therapy in a small group of patients with 
Hodgkin's Disease (completed last year), a 



NATIONAL CANCER INSTITUTE 



65 



larger study is now under way using a some- 
what different combination. Thus far, 35 pa- 
tients have been treated and although it is too 
early for final evaluation, it appears that more 
complete remissions are possible, particularly 
in the advanced stages of this disease, with this 
program than has been achieved before. The 
Radiation Branch has been extremely active in 
the treatment with radiation alone of patients 
with localized Hodgkin's Disease and of those 
in Stage Ill-a. The early results are very en- 
couraging and suggest that cure in some of the 
late stages as well as in the early stages is 
quite likely. 

Along with the programs in therapy, vari- 
ous other aspects of the lymphomas are being 
investigated. These include the infectious com- 
plications, immune mechanisms and the effect 
of anti-tumor agents on delayed hypersensi- 
tivity, antibody production, and lymphocyte 
transformation. Surprisingly, the studies to 
date do not support the previous reports that 
the immune mechanism of untreated patients 
with Hodgkin's Disease is uniformly depressed. 
Studies are underway to clarify our knowl- 
edge of immunity in anergic individuals. With 
the availability of large numbers of normal 
lymphocytes through the use of the NCI cell 
separator, certain fundamental studies are pos- 
sible. In addition, the demonstration that 
thoracic duct lymphocytes can circulate for as 
long as 9 days raises the possibility that those 
cells may be of value in anergic patients under 
certain circumstances. 

In addition to the large program in the 
treatment of lymphomas, the Solid Tumor 
Service has been vitally interested in the ther- 
apy of the blastic transformation of CML. This 
has been an utterly hopeless phase of the 
disease in contrast to the earlier phase in 
which there are a number of agents which can 
at least offer palliation to the patient. My- 
leran remains the drug of choice but a new 
drug, dibrom-mannitol appears promising and 
is being evaluated. In the blastic phase, the 
application of the POMP regimen or modifica- 
tions of it has not been remarkably successful 
but new agents including cytosine arabinoside 
and daunomycin are currently under study. 



"Life Island" 

Additional experience was gained in the use 
of the patient protection unit, the "Life Is- 
land." Patient accrual has been slow even 
though a second unit was obtained during the 
year. Ten patients have been treated in these 
units and several points appear obvious to 
date: (1) It is possible to maintain a patient 
for long periods of time (at least 3 months) 
in a relatively germ-free environment. (2) 
Under such circumstances and in spite of se- 
vere leukopenia, infection is not a major prob- 
lem even with serious toxicity. (3) Whether 
drug toxicity is reduced remains to be seen 
but thus far the impression is that it is some- 
what less even though large doses of drugs 
being employed. (4) Finally, none of the pa- 
tients experienced significant remissions of any 
duration in spite of the super-intensive ther- 
apy. The use of these units poses difficult prob- 
lems in nursing and patient care. Accordingly, 
plans for laminar flow rooms are in the early 
stages of formulation. Whatever the ultimate 
role of the germ-free approach will be, there 
is no question that this technique has impor- 
tant applications in cancer chemotherapy. 

Radiation Branch 

In addition to its important role in the 
studies described above, the Radiation Branch 
has achieved very encouraging results in ap- 
plying fractionated spaced total body irradia- 
tion of patients with chronic lymphocytic leu- 
kemia and lymphosarcoma. With this ap- 
proach, the selective destruction of abnormal 
cells apparently occurs while normal hema- 
topoietic elements are relatively untouched. 

Clinical Branch, Baltimore Public Health 
Service Hospital 

As indicated there were many administra- 
tive problems which had to be resolved in the 
relationship of this Branch to the NCI. How- 
ever, in spite of this, the Baltimore Branch 
made a major contribution to experimental 
chemotherapy effort. It was particularly active 
in the leukemias, lymphomas and testicular 
tumors. 



66 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Many studies were performed wholly or in 
part at the Baltimore Branch. It contributed 
a large number of patients both to the POMP 
protocol in the treatment of acute leukemia 
and the combination treatment (vincristine, 
nitrogen mustard, natulan, and prednisone) of 
Hodgkin's Disease. 

Cell Separation 

The joint effort with IBM to develop a ma- 
chine capable of separating granulocytes and 
platelets on a continuous flow in vivo basis 
saw some important progress during the year. 
With the development of a new model and a 
new centrifuge bowl design, it is now possible 
to separate 50% of both platelets and lympho- 
cytes passing through the machine. Attention 
is now being directed toward the concentra- 
tion of platelets which will permit their clinical 
use. 

A junior investigator who will work full 
time on the machine joined the program. Cur- 
rent studies are being done only on an in vivo 
basis using dogs primarily. Early results in 
the separation of granulocytes appear very 
promising in that yields are being obtained 
in the region of 35%. 

Conclusion 

It is apparent from the summary of the ac- 
tivities of the Clinical Trials area that the com- 
ing year should be a very exciting one. The 
organizational changes should permit a more 
intensive, efficient and intelligent effort in 
cancer chemotherapy and in the supporting 
programs. On the laboratory side important 
studies being carried out in leukocyte biochem- 
istry, leukocyte kinetics, cytogenetics, lympho- 
cyte transformation, immunity, electron mi- 
croscopy, tissue culture, etc., which are vital 
to the understanding of the malignant process. 
Generally, these have been undertaken because 
there were no similar studies elsewhere in the 
NIH and because the Clinical Trials area has 
the appropriate clinical material. 

The necessity for the diversity of studies 
within the Clinical Trials area makes it im- 
perative that additional senior personnel be 
acquired so that the various programs receive 



the direction of highly competent and trained 
individuals. A vigorous clinical program will 
be maintained. At the same time, in order to 
provide laboratory support for the clinical ac- 
tivities, at least two individuals with clinical 
interests as well as competence in such areas 
as biochemistry and immunology need to be 
recruited. Space, however, is a very serious 
problem, particularly in the Medicine Branch 
and makes it difficult to carry on existing pro- 
grams, let alone hire new investigators. 

With the studies now in progress, the com- 
ing year should bring significant advances in 
the treatment of acute leukemia and in Hodg- 
kin's Disease. There are strong indications 
that some of these patients will be cured. An 
important breakthrough appears imminent in 
the procurement of granulocytes and platelets 
in large numbers with the use of the NCI cell 
separator. Contracts with industrial firms will 
undoubtedly make it possible to provide these 
blood elements to other institutions on a wide 
scale. 

LABORATORY OF CHEMICAL 
PHARMACOLOGY 

The overall function of the OASDET in the 
search for chemical methods for the control of 
human neoplasms is the development of broad 
pharmacologic knowledge in laboratory ani- 
mals and in man of antitumor drugs. The de- 
gree of need for a comprehensive pharmacol- 
ogy program related to antitumor drugs de- 
pends in part upon the philosophy concerning 
the expected results of large scale screening 
programs. Extensive pharmacologic studies are 
not necessary if one subscribes to the notion 
that if enough compounds are screened long 
enough and hard enough a drug will be found, 
full blown, and fully effective which cures can- 
cer. The alternative, but not mutually exclusive 
notion, is that such a screening program will 
only provide leads to active antitumor com- 
pounds. Quantitative animal antitumor and 
host toxicity information concerning these 
compounds, and quantitative clinical toxicity 
and antitumor data, in conjunction with a 
thorough knowledge of the factors effecting 
the antineoplastic activity of such drugs (I 



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67 



consider all this pharmacology) is necessary 
to insure the development of chemical methods 
for the control of human neoplasms. 

While we are not involved in the primary- 
screening of compounds, we have responsibility 
in the field of experimental therapeutics, such 
as developing more accurate and reproducible 
systems for testing drugs, and in exploring 
more efficient means of treating tumor bear- 
ing animals by utilizing pharmacological in- 
formation. In the field of clinical chemother- 
apy, we are necessarily involved in the initial 
aspects at least of a new drug trial in terms of 
the clinical toxicology and pharmacology of a 
new drug. Information obtained from these 
clinical studies and studies in experimental an- 
imals is then used in the design and evalua- 
tion of the therapeutic trial. 

In the field of the pharmacology of chemo- 
therapeutic agents, we are responsible for de- 
veloping information on the factors, biological 
or chemical, which affect the antitumor ac- 
tivity of such drugs. 

The basic pharmacologic investigations in- 
clude a study of the absorption, tissue and cel- 
lular distribution, and excretion of these 
agents, and their metabolic fate. In addition, 
the biochemical mechanisms of action of anti- 
tumor drugs are investigated. Such data are of 
major importance in the design of the initial 
trial and the subsequent therapeutic trial in 
man. Another major area of research concerns 
the toxic effects of antitumor drugs. A 
thorough knowledge of the quantitative de- 
tails of the deleterious actions of antitumor 
drugs on normal cells and tissues is important 
in defining limitations in the therapeutic use 
of the drug. Of particular importance are quan- 
titative questions of extent or damage, rate of 
recovery and interval between dosing. 

In all these areas basic research is con- 
ducted, not only on the pharmacology of the 
specific drug, but also on the general princi- 
ples of pharmacology if these principles are 
not well known and understood. A recurring 
problem confronting us is that of comparative 
pharmacology. The development of any new 
drug from animal screening to clinical trial is 
an exercise in comparative pharmacology. The 
antitumor activity in the mouse and rat and 



the toxicity in mouse, rat and dog are used to 
attempt to predict activity and toxicity in man. 
The metabolism of a drug by mouse or rat 
tumor and normal cells is used as a basis for 
extrapolating to man. More information is 
needed comparing the pharmacology of such 
drugs in experimental animals and man. 

The following examples will illustrate cur- 
rent research activities relative to the afore- 
mentioned functions. The alkylating agents, for 
which the prototype is nitrogen mustard, are 
still important agents in the treatment of can- 
cer. Work this year has emphasized mecha- 
nisms of damage and repair of DNA by nitro- 
gen mustard with particular emphasis on cod ■ 
firming the hypothesis that crosslinking in tb 
eight position of the guanine moiety is a pr 
mary mechanism of action and further explor 
ing the possibility that nitrogen mustard mar 
also react with the phosphate groups. It was 
noted that DNA with at least two crosslinks 
between the chains, was still able to retain its 
transforming ability. This suggests that either 
a modest amount of alkylation does not impair 
the biological functions of this macromolecule, 
or that repair processes are available to neu- 
tralize the damage caused by crosslinking. A 
technique has been devised in which the alky- 
lated eight carbon in guanine may be specific- 
ally liberated as C0 2 under the proper condi- 
tions. This may allow a precise measure of the 
extent of crosslinking and for the development 
of firmer evidence that crosslinking in this 
position is critical to the action of mustard. 

The mechanism of action of hydroxyurea 
has yielded interesting and unexpected results. 
Hydroxyurea is degraded approximately 10- 
25% in vivo to urea. Evidence has now been de- 
veloped that this degradation is accomplished 
by a direct reduction through one unit of the 
oxidative cytochrome chain. This is similar to 
the reductive drug metabolic pathway present 
in liver microsomes; however, this reaction 
occurs in mitrochondria. It is interesting that 
this reduction of hydroxyurea to urea occurs 
in vitro in the presence of ferric ion. These 
results suggest that the mechanism of action 
of hydroxyurea is to act as a biological oxidiz- 
ing agent. This is consistent with the sugges- 
tion that hydroxyurea may damage cells by in- 



68 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



hibition of ribonucleotide reductase and sub- 
sequent nucleic acid synthesis. 

Work continues on the overall pharmacology 
of MIH, the methylhydrazine derivative active 
in lymphoma and Hodgkin's Disease. In its me- 
tabolic degradation, it is clear now that this 
compound goes through methylhydrazine. 
This can react with pyridoxal to yield pyri- 
doxal methylhydrazone, a vitamin B 6 antag- 
onist. There is also evidence that MIH admin- 
istration can, through other metabolic prod- 
ucts, exert monoamine oxidase inhibition. There 
is experimental and clinical evidence of cen- 
tral nervous system activity of MIH which is 
consistent with monoamine oxidase inhibition. 
In terms of its metabolism in mice, methane 
is one of the excretory products. The exact 
origin of this methane from the MIH mole- 
cule is as yet to be determined. 

Work continues on the nitrosourea deriva- 
tives. One difference between the metabolism of 
6fs-/?-chloroethylnitrosourea in man and lab- 
oratory animals appears to be that a much 
higher percentage of the BCNU remains in 
the body in man than in laboratory animals. 
In laboratory animals approximately 5% of 
the compound and its metabolites remain after 
24 hours; in man, approximately 25% remain 
after one week. The implications of this in 
terms of human toxicology are as yet unclear. 

One of the interesting new derivatives of 
nitrosourea synthesized by the Southern Re- 
search Institute group is cyclohexylfluorethyl 
nitrosourea. This is a highly active compound 
which shows less delayed toxicity in mice than 
BCNU. Preliminary toxicology studies indi- 
cated that in the rat there were unexpected 
acute deaths after the administration of cyclo- 
hexylfluorethyl nitrosourea. It was suggested 
that these deaths might be related to fluoro- 
acetate toxicity, arising from the degradation 
of the fluorethyl moiety of the nitrosourea 
compound. One biochemical consequence of 
fluoroacetate toxicity is an increase in the 
citric acid content of a number of organs, par- 
ticularly liver and kidney. It was shown that 
after an acutely toxic dose of cyclohexyl- 
fluoroethyl nitrosourea, citric acid concentra- 
tion in rat kidneys rose strikingly. This 
supports the notion that the fluoroethyl de- 



rivatives of nitrosourea might cause unusual 
acute toxicity related to the metabolism to 
fluoroacetate. 

Work is continued on the folic acid antag- 
onists. In an interesting combination of bio- 
chemistry and experimental therapeutics, ef- 
forts are now being made, using the high di- 
hydrofolic reductase containing strains of 
L1210, to assay for number of leukemia cells 
by assaying for folic reductase. In certain of 
these strains the very high enzyme activity 
will allow an excellent comparison of the direct 
decrease in enzyme activity and the direct kill 
of the cells to be compared against indirect 
methods relying on increase in life span or 
number of survivors. In other studies the abil- 
ity of kidney and choroid plexus to concen- 
trate methotrexate in vitro has been demon- 
strated. 

There are two main areas concerning the 
toxicology of antitumor drugs. In terms of re- 
search toxicology, we are attempting to define 
optimum dose schedules for a variety of the 
antitumor drugs which will minimize host tox- 
icity. In attempts to determine an ideal sched- 
ule with benefit with optimum relationship be- 
tween tumor damage and host survival, one 
approach is to search for that schedule which 
gives the least amount of host damage in the 
presence of large amounts of drug. For this we 
use the "priming dose study" in which a sub- 
lethal dose of the drug is given one day to a 
large group of animals. On subsequent days, 
portions of this large group of animals are 
assayed for the LD50 of this agent. With a 
compound like methotrexate, it can be shown 
that the LD50 is very low on days 2-4 after 
the administration of a sublethal dose. Subse- 
quently, on days 7-9, the LD50 is very high 
and the possibility exists, in fact, that the 
animals may be able to tolerate a higher dose 
of the drug. With a compound like cyclophos- 
phamide, there is only a modest decrease in 
the LD50 on days 2, 3, and 4, and a return to 
normal on subsequent days without rebound. 

Cytosine arabinoside appears to be similar 
to methotrexate in this respect. It is interest- 
ing to note that this rebound previously de- 
scribed for methotrexate, also occurs for 
cytosine arabinoside. This rebound can be seen 



NATIONAL CANCER INSTITUTE 



69 



after a single dose or infusion, but not after a 
course exceeding four or five days. One can 
visualize the bone marrow reserve which might 
be available for mobilization after a single 
dose may be damaged after repeated doses of 
the drug. 

A number of new agents were evaluated for 
their preclinical toxicology this year. These 
studies, vital to the development of new drugs, 
are summarized briefly in the appendix of this 
report. The increasing importance of quantita- 
tive toxicological studies has encouraged us to 
begin a program attempting to relate host tox- 
icity and experimental tumor damage to 
plasma concentrations of antitumor drugs. As 
mentioned above, the development of metho- 
dology and the beginning study of the physi- 
ological disposition of a variety of important 
anticancer drugs has begun. A group has been 
formed including representative of intramural 
NIH and all of the contractors who are involved 
in physiological disposition studies. This group 
will exchange information on techniques and 
methods for studying physiological disposition 
and will begin to pull together all the available 
data on the anticancer drugs and will initiate 
new areas in which studies should be per- 
formed. 

The technique of ventricular perfusion in the 
animal and its use as a research tool have 
been reported in the last few years, as well 
as the adaptation of this technique for clinical 
use in the treatment of intracranial malig- 
nancies. This technique has been further de- 
veloped in this past year. Doses of methotrexate 
up to approximately V2 to 1 liter of a 1 mgm/ 
millileter solution of methotrexate have been 
used in ventricular perfusion. This concentra- 
tion of methotrexate seems to be the maximum 
tolerated because occasionally transient con- 
vulsive phenomina occur at this concentration. 
Such concentration, of course, should be very 
effective in its antitumor effects. We have also 
studied the toxicology of 8-azaguanine and 
have begun a cautious clinical trial of this 
agent in ventricular perfusion studies. 8-aza- 
guanine is of particular interest because brain 
tumors are thought not to have the enzyme, 
guanase, that detoxifies this compound, but 
normal brain and most of the normal body 



tissues to have guanase and thus the quan- 
tities of 8-azaguanine which return to the blood 
stream after perfusion will be rapidly detoxi- 
fied. 

Three patients with meningeal leukemia who 
were refractory to intrathecal aminopterin 
have had complete remissions of their menin- 
geal leukemia after ventriculo lumbar per- 
fusion. The success of this sort of study en- 
couraged us to move on into the area of pri- 
mary intracranial neoplasms such as gliomas. 
In considering the chemotherapy of gliomas 
and other primary intracranial neoplasms, the 
need for an appropriate screening and test 
system became apparent. This is not available 
and therefore we began the process of develop- 
ing such a system, preferably in an animal 
large enough to use for pharmacology studies. 
Currently we are testing the Rous sarcoma 
virus in newborn puppies. This has been re- 
ported by Rabotti to cause ependymomas or 
gliomas. We are attempting, so far success- 
fully, to maintain the Kelly-O'Gara rhesus 
monkey hepatic carcinoma in sequential intra- 
cranial transplant in young rhesus monkeys. 
Although it is not a primary but a metastatic 
type tumor, its usefulness for pharmacologic 
studies seems clear. As a third alternative, we 
are exploring the YABA virus tumor inocu- 
lated intracranially. Some success was reported 
in the past by workers at Roswell Park; with 
more purified and highly potent virus there is 
reason to hope that this might yield a satisfac- 
tory brain tumor model. 

ETIOLOGY 

The Office of the Associate Scientific Direc- 
tor for Carcinogenesis was created by the re- 
organization of the Institute to plan and co- 
ordinate programmed research in experimental 
carcinogenesis. The Chemistry Section of the 
former Carcinogenesis Studies Branch was ele- 
vated to branch status (Chemistry Branch) to 
support a more comprehensive approach to the 
chemical biology of cancer. The remainder of 
the former Carcinogenesis Studies Branch 
(namely the Carcinogen Screening Section, the 
Bioassay Section, and the Cytogenetics and 
Cytology Section) have been regrouped into a 
branch (Biology Branch). 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The work of the Chemistry Branch, which 
emphasizes the fundamental nature of the in- 
teraction of carcinogenic agents with living 
systems in the induction of cancer at the host, 
tissue, cellular, and molecular levels, has pro- 
gressed in a most satisfactor manner during 
the fiscal year. The progress along specific lines 
of endeavor has been succinctly presented in 
the summary report of the Branch Chief; no 
effort will be made to reiterate at this point. It 
is important, however, to point out that the 
fundamental nature of the research has in- 
fluenced the Branch's scientists in their de- 
cision to perform their research activities in 
their NCI laboratory facilities rather than 
through collaborative relationships supported 
by contracts. As a consequence, the size of the 
research program that can be implemented 
is intimately related to the amount of space 
that can be made available to the Branch. Un- 
fortunately, during this prelude to the occu- 
pancy of the new cancer building, this Branch 
works in difficult circumstances of crowding 
and inadequate facilities, about which little 
can be done; the staff is to be commended 
highly for its productivity during this fiscal 
year. 

The Biology Branch has continued to main- 
tain collaborative studies, supported by con- 
tracts, in addition to its in-house activities. Its 
program has been aimed principally at the 
identification and characterization of known 
and suspected chemical carcinogens, especially 
those commonly occurring in the human en- 
vironment; efforts to improve bioassay meth- 
ods employed in the identification and char- 
acterization of chemical carcinogens with the 
aim of improving the relevance of animal data 
to the human situation; and the study of effects 
of known and potential carcinogens at the 
cytologic and genetic levels. The major findings 
emanating from the work during this year are 
stated in the report of the Biology Branch 
Chief, and will not be reiterated here. 

BIOLOGY BRANCH 

The Biology Branch, created by the reor- 
ganization, is composed of a Carcinogen 
Screening Section, a Bioassay Section, and a 
Cytogenetics and Cytology Section. Dr. Falk 



is the Acting Branch Chief, pending recruit- 
ment of another scientist for this position. 

The common thread linking the programs 
of the various sections in the Branch is ex- 
perimental carcinogenesis. The purpose of the 
experimental carcinogenesis in the Carcinogen 
Screening Section is the identification of en- 
vironmental carcinogens and the characteriza- 
tion of the metabolism and biological action of 
these carcinogens in vivo. The Bioassay Sec- 
tion is concerned with improvement of the 
methodology of identifying and characterizing 
chemical and other carcinogens in living sys- 
tems to the end that screening in animals and 
in vitro may be made more efficient and rele- 
vant to the human situation. The cytogenetics 
and Cytology Section emphasizes the morpho- 
logic and biochemical interaction of carcino- 
gens with the host at the cellular level. As 
will be readily appreciated, these sectional di- 
visions are somewhat arbitrary, the activities 
of the three Sections truly representing a con- 
tinuum rather than discreet items. 

The Acting Chief of the Branch, in addition 
to fulfilling his responsibilities as the Asso- 
ciate Scientific Director for Carcinogenesis, has 
maintained active in-house research programs 
described in Individual Project Report Nos. 
NCI-4600 and NCI-4601 concerned with the 
contribution of pesticides to carcinogenic bur- 
den in rats and pesticides synergists, and has 
been the responsible Project Officer on a spec- 
trum of contract-supported projects (#PH43- 
64-546, "Study of Air-borne Carcinogens by 
Means of Photodynamic Bioassay"; #PH43- 
64-45, "The Co-Carcinogenicity of Aliphatic 
Hydrocarbons and Organic Disulfides"; 
#PH43-65-1036, "Carcinogenesis: Toxins in 
Foodstuffs"; # PH43-64-933, "Studies on 
Mucous Flow in the Intact Mammal Exposed 
to Tobacco Smoke"; #PH43-63-1165, "Oxi- 
dation Products of Fats"; #PH43-64-1158, 
"Piperonyl Butoxide as Carcinogen or Co- 
Carcinogen"; #PH43-64-567, "The Prepara- 
tion of Products Related to Dibenz (ah) An- 
thracene and Benzo(a)Pyrene"; #PH43-64- 
938, "Tumor-Promoting Principles of Tobacco 
and Tobacco Smoke"; # PH43-64-865, "In- 
vestigation of Potential Anti-Radiation and 
anti-Neoplastic Compounds"; #PH43-64- 



NATIONAL CANCER INSTITUTE 



71 



504, "Biochemical and Morphological Compo- 
nents of Hepatic Carcinogenesis; #PH43-66- 
508, "Potential Carcinogenicity of Cancer Che- 
otherapeutic Drugs"; #PH43-65-1000, "Re- 
search on Free Radicals and Alkylating Agents 
in Tobacco Smoke"; #PH43-65-1029, "Car- 
cinogenic Mycotoxins"). In addition, he has 
had a major share of the responsibility for the 
contract relationship with the Chicago Medical 
School (#PH43-65-67), under which a large- 
scale carcinogen screening program is main- 
tained and research on the fundamentals of 
cancer induction mechanisms is pursued. 

The pesticides carcinogenicity studies being 
conducted under contract by the Bionetics Re- 
search Laboratories, with close supervision 
by the Biology Branch scientists, has been 
progressing in a highly satisfactoiy manner. 
An exemplary number of valuable contribu- 
tions to the literature have been made with 
respect to pesticides chemistry; the long-term 
carcinogenicity experiments will be completed 
during the ensuing six months. The information 
to be obtained from these studies has been 
long awaited with great interest. The contract- 
supported research project on the carcinogeni- 
city of oxidized lipids, being conducted at Gen- 
eral Foods Corporation, has been augmented 
to include the determination in animals of car- 
cinogenicity of these lipid products. A new 
contract-supported research project has been 
initiated, in collaboration with the CCNSC, at 
the University of Pittsburgh for the determina- 
tion of the carcinogenicity of a number of 
chemo-therapeutic agents employed in clinical 
medicine. A complementary study of the car- 
cinogenicity of nitrofuran derivatives is being 
negotiated with the University of Wisconsin. 
The detailed descriptions of the established 
projects will be found in the contract narra- 
tives of this Branch. 

The Acting Branch Chief has been a prime 
force in the effort to obtain a collaborative 
relationship with Dr. Norton Nelson's group 
at New York University, for experimental car- 
cinogenesis involving asbestos products, poly- 
urethane foam products, peroxides, and epox- 
ides, and research into the carcinogenic and 
co-carcinogenic constituents of tobacco and to- 
bacco smoke. 



The Head of the Carcinogen Screening Sec- 
tion, while serving as an Assistant Project 
Officer on the Chicago Medical School contract, 
has been responsible for a number of other 
contract-supported carcinogen screening activi- 
ties and has maintained his own active in- 
house research program. These screening ac- 
tivities have resulted in the identification of 
about fifteen hitherto unrecognized carcino- 
gens. The Section's in-house research has made 
important contributions to the literature on 
the effects of androgens and estrogens on the 
susceptibility of rat liver to chemical carcino- 
gens; has developed a new system for the sepa- 
ration of urinary metabolites of carcinogenic 
aromatic amines; has described the forms in 
which carcinogenic amines and their metabo- 
lites circulate in the blood and transfer from 
one tissue compartment to another; has shown 
some of the effects of pituitary and adrenal 
hormones on the metabolism of N-hydroxy-N- 
2-fluorenylacetamide to increase the level of 
the proximate carcinogen; has shown that 
some carcinogens combine with existing cel- 
lular and molecular targets rather than with 
the same targets during their periods of syn- 
thesis; and has demonstrated the competitive 
inhibition of binding of carcinogens at the 
target, using chloramphenicol. The utility of a 
standardized means of large-scale chemical car- 
cinogen screening and further progress on the 
synthesis and structural studies of naturally 
occurring carcinogens, such as aflatoxins, have 
been accomplished by contract-supported proj- 
ects. Members of the staff of this Section au- 
thored a number of review articles intended 
for wide circulation to alert chemists to the 
potential carcinogenic effects of the products 
of industry. 

The Bioassay Section has maintained con- 
tract-supported projects for the improvement 
of the methodology of carcinogen screening, 
while conducting its own research on the inhi- 
bition or anti-tumorigenic action of actinomy- 
cin-D. The screening methodology studies have 
emphasized the utilization of the mammary fat 
pad technique of DeOme and Faulkin, and 
the exploitation of the unusual susceptibility 
and promptness of response to carcinogenic 



72 



ANNUAL EEVIEW OF INTRAMURAL RESEARCH 



stimuli by animals in their neonatal period. 
Efforts are being made to evalute the useful- 
ness of animals born in particularly immature 
states, such as marsupials. More fundamental 
studies have demonstrated an anti-tumorigenic 
effect in skin carcinogenesis with 90/xg of 
actinomycin. In addition, actinomycin (1 to 
90/xg) inhibited RNA synthesis in skin, whereas 
initiating amounts of DMBA produced a 
slight inhibition of RNA synthesis. This in- 
hibition occurred shortly after treatment with 
DMBA and was transitory. The inhibition of 
RNA synthesis by actinomycin occurred some- 
what later and was of a more lasting nature. 
The antitumorigenic effect of 90/xg of acti- 
nomycin was accompanied by an increase in 
protein synthesis, a change in mitotic index 
for interfollicular epidermis, and hyperplasia. 
Skin tumorigenesis was also inhibited by 
maleic anhydride, N-ethyl maleimide, and iodo- 
acetamide, agents which combine readily with 
-SH groups. These findings support the hypo- 
thesis that DMBA skin tumorigenesis may in- 
volve interaction with these -SH groups. Anti- 
tumorigenesis is not seen when DMBA is ap- 
plied 2 to 8 weeks after treatment with maleic 
anhydride, but is noted when this period is 
reduced to one week. Treatment with maleic 
anhydride for ten weeks produced inhibition, 
while treatment for one week was ineffective. 
Actinomycin was ineffective in inhibiting lung 
tumorigenesis even when administered at a 
high dose, despite a transient inhibition of 
RNA synthesis. Studies of DNA metabolism 
have been pursued, and interesting findings 
reported in the Individual Project Reports for 
this Section. A fascinating and important ob- 
servation has been the stronger carcinogenic 
stimulus by smooth-surfaced plastic implants 
than by those with rough surfaces. This find- 
ing may have some significance in the use of 
plastic prostheses in human beings. 

The transfer of Dr. Klein to Extramural Ac- 
tivities, NCI, will necessitate a recruiting ac- 
tion for the position of Head of the Bioassay 
Section. Dr. Klein's responsibilities as a Proj- 
ect Officer will be distributed among other 
members of the Branch. 

The Cytogenetics and Cytology Section is 
studying the quantitative effects of carcino- 



gens, employing models designed primarily to 
investigate somatic viruses. The systems in use 
include microbial, cell, and organ cultures and 
intact animals. Thus, this Section tends to 
bridge both the biological and chemical ap- 
proaches to carcinogenesis. A critical review 
of both the cancer and teratology literature 
made by the Section has resulted in the con- 
cept that the same environmental insult may 
be responsible for either cancer or malforma- 
tions or both. In this area, it has been demon- 
strated that important carcinogens, such as 
aflatoxins, uracil mustard, and urethan, all pro- 
duce malformations; the severity of the mal- 
formations being dependent upon the dose ad- 
ministered. The extent of the parallelism be- 
tween carcinogenic and teratogenic activity is 
being explored by the use of analogues and 
inhibitors of carcinogens. The effects of car- 
cinogens at the cellular level in terms of drug 
resistance and neoplastic transformation are 
under way using mouse and hamster material. 
Heart tissue, which is rarely the site of pri- 
mary malignancy, has been shown to undergo 
spontaneous neoplastic transformation result- 
ing in mesenchymal tumors. In addition, tissue 
culture cells underwent chromosomal changes 
consistent with an evolutionary pattern of 
genetic readjustment. Hamster embryo cells 
when exposed to carcinogenic polycyclic hydro- 
carbons underwent a series of morphological 
changes, including loss of contact inhibition, 
inhibition of mitosis, and death of many cells. 
Cells grown in the presence of the carcinogen 
resulted in permanent cell strains which pro- 
duce tumors when inoculated into hamsters. 
Transformed cells were resistant to the ex- 
posure of additional carcinogens while control 
cells exhibited toxicity. Current experiments 
are aimed at determining the possibility of 
selection of spontaneously transformed cells 
by the carcinogen, the acceleration of the car- 
cinogenesis process by the chemical or the ac- 
tual induction of the change by the carcinogen. 
Alkylating agents have been studied using spe- 
cific synthetic messenger ribonucleic acid 
(RNA) and using tissues of mice treated with 
uracil mustard. The synthetic messenger was 
inactivated by alkylating agents by either es- 
terification of chain terminal phosphate or 



NATIONAL CANCER INSTITUTE 



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cross-linking strains of RNA by forming di- 
guanyl derivatives. The administration of a 
high dose of uracil mustard to Strain A mice 
caused a marked inhibition of DNA synthesis 
in lung, liver, and kidney. Inhibition of total 
RNA synthesis and nuclear RNA synthesis 
was also observed at relatively high dosages 
of uracil mustard in the three tissues studied. 
Additional descriptions of these studies are 
contained in the Individual Project Reports of 
this Section and in the report on the Temple 
University contract (#PH43-66-68) with 
Dr. Daniel Swern. 

CHEMISTRY BRANCH 

The program of the Chemistry Branch is de- 
signed to clarify the fundamental nature of the 
interaction of carinogenic agents with living 
systems in the induction of cancer at the host, 
tissue, cellular, and molecular levels, and to in- 
vestigate the interaction of certain environ- 
mental agents with functioning biological sys- 
tems. The biochemical alterations in carcino- 
genesis and in the resultant tumor tissue are 
expressed as a number of phenotypic changes 
which include altered enzyme profile, altered 
responsiveness to environment, increased 
growth, loss of contact inhibition, and loss of 
specialized function. Since the phenotypic 
changes are heritable, they presumably reflect 
alterations in gene function, either at the ge- 
netic transcription level or at the translational 
level in the cytoplasm. The Chemistry Branch 
studies the biochemical nature of the factors 
controlling gene activity and messenger RNA 
translation, as well as the chemical nature of 
the genetic factors characterizing susceptibil- 
ity to carcinogenesis. Studies are made on the 
effects of carcinogens of various types, includ- 
ing chemical and viruses on geneaction sys- 
tems. The studies are performed on both the 
nature of the chemical and physical interaction 
of carcinogenic agents with various cellular 
components, and the effect of their interaction 
on the functional integrity of cellular control 
systems. 

Studies designed to elucidate the biochemi- 
cal nature and the time required for the initia- 
tion of skin tumorigenesis are in progress. One 



definitive result has shown that the time re- 
quired for the initiation of skin tumorigenesis 
is of short duration, of the order of 1 to 2 
days. In addition, the initiation of skin tumori- 
genesis was found to depend on functioning 
gene activity and potent carcinogenic agents 
did not elicit initiation of tumorigenesis when 
the inhibitor of gene activity, actinomycin D 
was present. 

In related studies, actinomycin D was found 
to decrease the amount of carcinogen bound 
to skin DNA by 35% and not to affect the 
amount of carcinogen bound to total skin pro- 
tein. In other studies actinomycin D as well 
as the carcinogen, 7,12-dimethylbenzanthra- 
cene (DMBA), were both found to inhibit 
DNA and RNA synthesis. 

Investigations on the interaction of liver 
carcinogens with cellular macromolecules have 
demonstrated that three potent liver carcino- 
gens, N-hydroxy-AAF, 3'-methyl-DAB, and 
aflatoxin B 1( interact with DNA and cause a 
reduction in the RNA to DNA ratio of puri- 
fied liver nuclei. In addition, these agents were 
found to inhibit the incorporation of radioac- 
tive precursors into nuclear RNA. Thus these 
carcinogenic agents are altering gene activity 
at the level of RNA synthesis. 

Investigations on the mechanism of malig- 
nant transformation by Rous sarcoma virus 
(RSV) has shown that the initial stage of the 
transformation process requires DNA synthe- 
sis, and that subsequent stages allowing for the 
growth of RSV require the continuous partici- 
pation of cellurlar DNA. These important re- 
sults have clearly increased our understanding 
of the nature of malignant transformation by 
Rous sarcoma virus. As a corollary to these 
studies techniques have been developed for the 
growth of Rous sarcoma virus in liquid spin- 
ner cultures and in cells suspended in agar. 
The ability of cells to grow under these con- 
ditions requires their prior infection by Rous 
sarcoma virus. These techniques will permit 
the large scale production of Rous sarcoma 
virus. 

Investigation of vesicular stomatitis virus 
infection has shown that it induces in infected 
cells a new enzyme capable of synthesizing 
RNA. This enzyme did not appear in cells in- 



74 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



fected with Rous sarcoma virus or Rous-asso- 
ciated virus. 

Methods have been developed for the isola- 
tion of macromolecular components of liver 
chromatin under conditions in which the 
physiological relationship between these com- 
ponents is minimally disturbed. Using- some of 
these techniques, DNA molecules with a mole- 
cular weight of 3 times 10 8 daltons have been 
isolated from chick embryo. The distribution in 
DNA size was found to be unimodal, which is 
unlike the size distributions of metaphase chro- 
mosomes in this organism. An accurate deter- 
mination of DNA size will permit the investi- 
gation of the relationship between DNA size 
and carcinogenesis. In other studies DNA from 
mouse liver nuclei and mitochondria has been 
isolated and examined in respect to molecular 
weight and buoyant density. These studies 
have found that the minor component of mouse 
liver DNA, the satellite band, comprises about 
10% of the nuclear DNA and is not found in 
mitochondrial DNA preparations. 

The molecular basis for the mutagenic action 
of hydroxylamine, nitrous acid, formaldehyde, 
and the carcinogen, N-methyl-N-nitroso-ure- 
than has been investigated. Hydroxylamine 
acts by altering the coding properties of de- 
oxycytidylic acid from guanylic acid incorpo- 
ration to adenylic acid incorporation. Formal- 
dehyde causes inactivation of the template 
properties of polymers, and the carcinogenic 
N-methyl-N-nitroso-urethan alters several 
parameters of template activity which are now 
being studied. 

Studies on the removal of messenger RNA- 
like activity from rat liver microsomes have 
found that this activity can be removed with- 
out disturbing the capability of microsomes 
to incporporate amino acids in the presence of 
synthetic messenger RNA. Indeed the removal 
of endogenous messenger RNA increases the 
sensitivity of microsomes to added synthetic 
messenger RNA in respect to both the ability 
to bind synthetic messenger RNA and the ac- 
tivity of the synthetic messenger RNA in di- 
recting amino acid incorporation. 

The effects of carcinogens, non-carcinogenic 
drugs, or environmental chemicals on the in- 
duction of specific drug metabolizing enzymes, 



and the mechanisms of this induction have 
been under study. The induced enzymes control 
to a significant extent the duration of drug ac- 
tion; their altered activity may be responsible 
for often-observed altered drug resistance and 
susceptibility. In addition, these hazards of 
drugs and environmental agents are capable 
of altering normal metabolism. Thus, pheno- 
barbital was found to increase the messenger 
RNA content of microsomes. It also increased 
the sensitivity of microsomes, but not ribo- 
somes, to the addition of synthetic messenger 
RNA. Phenobarbital also increases the endo- 
plasmic reticulum of the cell. Investigations 
have also been made of the effect of some of 
these agents on the genetic apparatus, and in 
particular, on RNA polymerase activity of the 
nucleus. 

Methods, utilizing the zonal centrifuge, are 
being developed for the preparation and iso- 
lation of macromolecules and cellular and sub- 
cellular fractions of biological systems. Separa- 
tions have been made of E. coli RNA, E. coll 
ribosomes, rat and mouse liver ribosomes, mi- 
crosomes, mitochondria, and whole cells. In ad- 
dition, preliminary work suggests the feasibil- 
ity of separating cell membranes and nerve 
endings. These techniques will greatly enhance 
the ability to isolate large amounts of cellular 
components needed for biochemical investiga- 
tions. 

An extensive electron microscope study of 
transformed tissue culture cells is in progress. 
This study has revealed that the so-called non- 
producer cells release morphologically complete 
but noninfectious viral particles. In addition, 
certain surface morphology appears altered in 
the transformed cells. In addition, many of the 
subcellular materials separated by the zonal 
centrifuge are being examined by electronc mi- 
croscopy. The electron microscopic pictures 
have contributed a semi-quantitative estimate 
of the number and kinds of riobosomes of vary- 
ing polymeric units. The distribution of these 
subcellular particles in the tumor and normal 
cells is being compared. The electron micro- 
scope has also been applied to the study of the 
epithelial cells of the bronchial tree in animals 
exposed to air pollutants. The electron micro- 
scopy study has provided evidence for a differ- 



NATIONAL CANCER INSTITUTE 



75 



ent degree of response and rate of recovery in 
the epithelial cells at different levels of the 
bronchial tree. 

Studies in the Section on Proteins have in 
the past year included the use of zone electro- 
phoresis in acrylamide gels as an analytical 
and preparative technique, studies of serum 
proteins in man and rodents, correlations be- 
tween serum haptoglobin type and disease, and 
studies on the preparation and properties of 
haptoglobin type 1-1 in human serum. 

A study of several hundred normal human 
serum specimens has confirmed the high reso- 
lution of "disc" electrophoresis in acrylamide 
gels. The major polymorphic systems demon- 
strated by such analyses are the group-specific 
component, transferrin and haptoglobin. Most 
variations between individuals could be attrib- 
uted to these systems. Other variations also 
exist, but could not be similarly explained. 

The high resolving power of such gels has 
been adapted to preparative purposes with ex- 
cellent results in the case of hemoglobin and 
haptoglobin type 1-1. Both these proteins have 
been isolated in highly purified form, with less 
than 1% detectable impurities. 

The interaction of haptoglobin and hemoglo- 
bin has been studied with this purified mate- 
rial. This interaction passes through an inter- 
mediate in which one-half molecule of hemo- 
globin reacts with one molecule of haptoglobin. 
This complex may add another one-half mole- 
cule of hemoglobin to form a fully saturated 
complex. A detailed analysis of this reaction 
was possible. 

An analysis of haptoglobin types in mice has 
shown that inbred strains differ with respect 
to whether they have haptoglobin. AKR and 
C 3 H mice, although normally without serum 
haptoglobin, are easily induced to produce it. 
Variations between mouse strains in this con- 
nection may be related to ease of "spontan- 
eous" tumor formation. 

Further data on haptoglobin types in hu- 
man keukemia confirm that such patients have 
an excess of the Hp 1 gene, over normal groups. 
Similar, but less marked, differences are found 
between control groups and female patients 
with carcinoma of the breast and male patients 



with carcinoma of the lung. A report showing 
an excess of Hp 1 in patients with juvenile 
rheumatoid arthritis could not be confirmed. 
An improved haptoglobin typing method was 
devised and used in these studies. 

VIRAL ONCOLOGY 

Activity in the tumor-virus program of NCI 
was further increased during the past fiscal 
year. An increase during the preceding year 
had brought about a split of the Laboratory of 
Viral Oncology and the creation of an addi- 
tional laboratory, the Laboratory of Viral Car- 
cinogenesis as well as of the Office of the 
Associate Scientific Director of Viral Oncology 
to coordinate tumor-virus research within the 
two laboratories. With the reorganization of 
NCI during the past fiscal year, this office and 
its constituent laboratories were transferred to 
the Office of the Scientific Director for Etiol- 
ogy, where they were consolidated with the 
tumor-virus program already in that Office 
within the branch previously designated as the 
Virology Research Resources Branch. The new 
designations of these laboratories and 
branches and their respective Branch Chiefs, 
are as follows: (1) Viral Biology Branch, Dr. 
A. J. Dalton, Chief; (2) Viral Carcinogenesis 
Branch, Dr. R. E. Stevenson, Chief; and (3) 
Viral Leukemia and Lymphoma Branch, Dr. 
F. J. Rauscher, Jr., Chief. 

The Viral Carcinogenesis Branch, which su- 
perseded the former Virology Research Re- 
sources Branch, continued its functions with 
respect to the development and management 
of resources essential to tumor-virus research, 
and assumed additional responsibility for di- 
rect program activities in the field of naked 
DNA tumor viruses. Pending completion of the 
new NCI building and the availability of lab- 
oratory space, the NCI, through the Viral Car- 
cinogenesis Branch, will embrace an intramu- 
ral research program on these DNA tumor 
viruses for the first time. Key staff scientists 
who will participate in this research as well as 
in other program activities have already been 
recruited. This Addition of scientific personnel 
and expertise has made possible the activation 
under contract of new major activities in sup- 
port of cancer- virus research, such as: (1) the 



76 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



viral diagnostic laboratory which will identify 
known agents and differentiate new agents 
turned up in the search for human cancer vi- 
ruses by scientists participating in special pro- 
grams of NCI; and (2) research on the devel- 
opment and production of simian virus re- 
agents, which will be critical for monitoring for 
and identification of contaminant simian vi- 
ruses if a human leukemia or other cancer virus 
is found which is transmissible in these pri- 
mate species. 

The oncogenic naked DNA viruses, which in- 
clude 5 of the human adeno-viruses, were dis- 
covered by virologists employing the tissue 
culture techniques of classical virology, and 
their intensive investigation has been pursued 
primarily in general virology laboratories. Up 
until recently the principal activity of the NCI 
in this area has resided in support of the com- 
munity of virologists engaged in oncological in- 
vestigations, through its virology research re- 
sources program. Developments during the past 
two years, and particularly during the last fis- 
cal year have revealed neoantigens in tumor 
cells of hamsters and other rodents induced by 
human adeno and other oncogenic naked DNA 
viruses, although infectious DNA virus is not 
reproduced in the oncogenic interactions. 

At the joint suggestion of Doctors Albert 
Sabin and Robert Huebner, an ad hoc group of 
leading virologists concerned with this problem 
was brought together for several discussions 
during the past year, under the sponsorship of 
the Viral Carcinogenesis Branch. The question 
was examined whether the time was ripe for a 
joint programmed activity in search of neoan- 
tigens in human cancers that are characteris- 
tic for certain known human viruses, including 
the adenoviruses. Although the time seems near 
when such studies might be initiated, it was 
concluded that refinements in existing tech- 
niques must be made before a satisfactory 
study can be launched. This burgeoning collec- 
tive effort may presage a future major program 
activity of NCI. 

The Viral Leukemia and Lymphoma Branch 
and the Viral Biology Branch have continued 
their singular as well as their joint activities 
involving the longer known membrane-bound 
tumor viruses of the types associated with nat- 



urally prevalent and transmissible neoplasms 
of animals. These include: (1) the C type RNA 
viruses associated with avian and murine leu- 
kemia and related neoplasias, and which bud 
from cytoplasmic membranes; and (2) "herpes- 
like" particles such as the Lucke frog kidney 
carcinoma virus which matures in the nucleus, 
and probably contains DNA. Viruses of both of 
these types are reproduced during, and remain 
associated with the overt neoplastic disease 
which they induce. 

In studies directed toward speeding the 
search for comparable agents in human leu- 
kemia and lymphoma, scientists of the Viral 
Leukemia and Lymphoma Branch have adapted 
two additional highly sensitive immunological 
techniques to the detection of murine leukemia 
virus antigen, and are now investigating appli- 
cations to the human problem along the same 
lines as previously reported for the fluorescent 
antibody technique (i.e., the detection of spe- 
cific, leukemia associated, antigens and/or anti- 
bodies which could be of viral derivation). The 
additional techniques, which have proven more 
sensitive than the fluorescent antibody tech- 
nique in murine systems, are: (a) the micro- 
Ouchterlony (double diffusion in gels) precip- 
itin reaction; and (b) specific hemagglutination 
of antigen-coated tanned erythrocytes. Consid- 
erable progress has also been made in the con- 
trolled degradation of murine leukemia viral 
particles into morphological subunits, and puri- 
fication of the subunits by density gradient 
centrifugation. 

The Viral Biology Branch continues to play a 
leading role in the search for viral etiological 
agents associated with human leukemia and 
lymphoma, using particularly the methods of 
electron mircroscopy and tissue culture. In col- 
laboration with clinical staff members 141 
plasma specimens from 120 separate cases of 
leukemia were studied during the past year. Of 
these, about 13% were found to contain virus- 
like particles resembling the C type virus parti- 
cles of animal leukemias. This is approximately 
the same proportion of positive specimens ob- 
served in previously reported studies. The re- 
sults are consistent with those obtained in neo- 
plastic diseases of animals induced with low 



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doses of RNA tumor viruses or in the naturally- 
occurring animal diseases. 

Membrane-bound virus particles of the 
"herpes" type have been observed by electron 
microscopy in 3 of 4 additional cell lines of 
Burkitt lymphoma sent to Dr. Manaker from 
Nigeria by Dr. Pulvertaft (University of Iba- 
dan). This brings to 9 the number of estab- 
lished cell lines of Burkitt lymphoma that have 
been investigated in several laboratories for 
the presence of virus-like particles. The 
"herpes-like" particle has been observed in 8 of 
the 9 cell lines. Similar particles were also ob- 
served in tissue culture cells from: (1) an in- 
duced myeloid leukemia of a monkey (original 
specimen supplied by Dr. Margaret Kelly of the 
Chemotherapy area of NCI); and (2) a human 
patient with chronic myeloid leukemia. The lat- 
ter observation brings to 5 the number of es- 
tablished cell lines of human leukemia that 
have shown "herpes-like" particles, out of a 
total of 12 investigated by participants in the 
Special Virus-Leukemia Program. 

Whether the "herpes-like" agent is a ubiqui- 
tous "passenger" virus, or whether it is a se- 
rious contender for an etiological role is not 
yet clear. It cannot be propagated in any cell 
line thus far tried, other than the explanted 
malignant cells in which it is found naturally. 
It shows no antigenic relationship to other 
members of the herpes group, or any other 
known virus thus far compared. 

In addition to basic research on membrane- 
bound tumor viruses, and experimental partici- 
pation in the search for counterpart human 
agents, members of the Viral Biological Branch 
are Chairmen of two major Segments and Vice 
Chairmen of two Segments of the Special Virus 
Leukemia Program. They also serve as Project 
Officers and supervisors of developmental re- 
search under contract amounting to approxi- 
mately $5 million. 

VIRAL BIOLOGY BRANCH 

Electron Microscopy Section 

Highlights of the results of collaborative 
studies on animals in which electron micro- 
scopy played an important role include the 



demonstration that the etiologic agent of Mo- 
loney sarcoma is indistinguishable morphologi- 
cally from the murine leukemia virus; the vis- 
ualization of the process of replication of 
avian-type viruses in brain tumors of the dog, 
induced by Rous sarcoma virus; and the dem- 
onstration of the presence of particles in tissue 
culture cells derived from the buffy coat of a 
monkey with chronic myelogenous leukemia 
having the same morphology as those associ- 
ated with tissue culture isolates from Burkitt 
lymphoma. 

In studies on human material, it has been 
found that the virus particles associated with 
Burkitt lymphoma differ from known herpes 
type viruses by the acquisition of a granular 
coat at some time during maturation. Also 
worth of note is the finding of Burkitt type 
particles in tissue culture cells derived from 
the buffy coat from a human case of chronic 
myeologenous leukemia. These findings may 
still be explained on the basis of passenger 
virus, but the presence of these particles in 8 
out of 9 Burkitt lymphoma isolates and in iso- 
lates from 5 human cases of myeologenous leu- 
kemia (including the 4 Roswell Park isolates) 
suggests that something more than simple co- 
incidence is involved. 

In the screening program, 19 out of 141 
plasmas from leukemic patients were found pos- 
itive for virus-like particles, an average of 
something more than 13%. A shift in emphasis 
is planned for this work with increased con- 
centration on lymph node biopsy material and 
less on plasma pellets. 

Microbiology Section 

Work in this Section has included the suc- 
cessful propagation of Burkitt lymphoma iso- 
lates, EB-1, 2, and 3 of Epstein and the 
Jiyoye, Kude, Ogun, and Ragi lines of Pulver- 
taft. Modification of the culture media and 
other experimental interventions were at- 
tempted with the aim of increasing virus yield. 

In addition the crossing of species barriers 
has been demonstrated by tumor induction fol- 
lowing intracranial inoculation of high titer 
Rous sarcoma virus into rabbits, guinea pigs, 
hamsters, cats and dogs. The common forms of 
tumors have been gliomas and meningiomas, 



78 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



although the tumors in guinea pigs have been 
difficult to classify. 

An experiment involving the feeding of mos- 
quitoes (Aedes egypti), first on Burkitt tissue 
culture cells, then on a monkey resulted even- 
tually in a modified peripheral blood picture 
and enlarged lymph nodes in the monkey. There 
has been no evidence of the development of the 
leukemic state, but this experiment should be 
followed up. 

It should be noted that the head of the Sec- 
tion on Microbiology spends fully two-thirds of 
his working hours on administrative matters 
connected with the SVLP. 

Virus Studies Section 

Extracts from the SL-1 isolate from Burkitt 
lymphoma suspended in 5% dimethyl sulphox- 
ide (DMSO) inoculated intracranially into 
newborn hamsters induced CNS symptoms, 
eventually as early as 5 or 6 days after inocu- 
lation. Members of the staff at the Pfizer Lab- 
oratories, Maywood, N.J., have confirmed these 
findings for material obtained from the Virus 
Studies Section but have been unable to confirm 
them starting with fresh homogenates of SL- 
1 or EB-2 Burkitt isolates. To date the virus 
which undoubtedly is present has not been vis- 
ualized and it is important to determine 
whether it is of human origin or is a hamster 
virus activated by the treatment. 

Virus Leukemia Section 

Studies have continued on the virus induced 
murine sarcoma. Histopathological analysis in- 
dicates that it is a rhabdomyosarcoma with 
cross striation sometimes apparent in the 
larger of the two cell types. The large cells when 
prepared as whole mounts are very similar in 
appearance to cells present in human rhabdo- 
myosarcoma. 

Attempts to pass this virus into cortisone- 
treated rats have been successful with tumors 
similar to those induced in mice appearing 
within 1 to 2 months. Continued passage in 
rats has resulted in death within 24-36 hours 
after inoculation. This phenomenon is at pres- 
ent under study. 



An analysis of the results of the examination 
of human leukemic plasma pellets has demon- 
strated that there is a direct correlation be- 
tween high white cell counts and plasma pel- 
lets positive ofr virus-like particles. No other 
correlation has been found. 

It has been reported previously that an agent 
present in the plasma ceil tumor MPC-2 in- 
duces reticulum cell sarcomas. Since these tu- 
mors are very similar to Hodgkins sarcoma in 
man, it would be important to determine if they 
are consistently induced by a viral agent. Stud- 
ies under way suggest this as a possibility. In 
other experiments an agent has been demon- 
strated to be present in Sarcoma-37 which in- 
duces bone lesions in mice. These lesions are 
similar to the osteopetrosis induced in chick- 
ens by viruses of the avian leukosis complex. 
Evidence to date suggests that the agent from 
S-37 is the same as or closely related to the 
Moloney leukemia agent. This in turn suggests 
the existence of an important and fundamental 
similarity between the range of tumor types 
induced by viruses of the avian leukosis com- 
plex and viruses of the murine leukemia fam- 
ily. Improved transparent chambers were in- 
volved in the development of the information 
obtained from the last two studies. It should be 
noted that the head of the Virus Leukemia Sec- 
tion spends fully two-thirds of his working 
hours on administrative matters concerned 
with the SALE segment of the SVLP. 

VIRAL LEUKEMIA AND LYMPHOMA 
BRANCH 

Introduction 

The Viral Leukemia and Lymphoma Branch 
was organized and officially approved in De- 
cember of 1965 as a part of the overall reor- 
ganization of the National Cancer Institute. 
The personnel and intellectual nucleus of this 
Branch were derived almost entirely from the 
Laboratory of Viral Oncology. Dr. W. Ray 
Bryan, as Chief of this Laboratory, assembled 
and guided a group whose outstanding compe- 
tence and productivity became internationally 
acknowledged. Organizationally the Branch as 
proposed will include sections on Comparative 
Viral Oncology, Immunology, Molecular Virol- 



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79 



ogy, Ultrastructural Studies, and on Virus and 
Disease Modification. Laboratory personnel wh 
are currently functioning- as Section Heads 
are: Dr. Mary Fink, Immunology Section, with 
8 support personnel; Dr. Timothy E. O'Connor, 
Molecular Virology Section, with 5 support 
personnel; Dr. Robert F. Zeigel, Ultrastruc- 
tural Studies Section, with 4 support person- 
nel. In addition, the transfer of Dr. Michael A. 
Chirigos and 4 support personnel from the 
Chemotherapy area to the Viral Leukemia and 
ymphoma Branch will be consummated with- 
in this fiscal year. Dr. Chirigos will head the 
Virus and Disease Modification Section. Re- 
cruitment actions of key scientists are cur- 
rently being processed. These and other actions 
and activities are being conducted in a manner 
that will take advantage of currently available 
space and equipment and in a manner that will 
allow a minimum of delay between the current 
level of activity and the expanded level which 
will be possible as soon as the new cancer 
building (Building 37) and the emergency vi- 
rus isolation building are completed (approxi- 
mately January 1968). 

The principal activities conducted within the 
Branch during fiscal year 1966 are discussed 
briefly according to the following categories: 
(a) research and other activities conducted 
within the Branch, and (b) Special Virus- 
Leukemia Program. 

Research and Other Activities Conducted Within 
the Branch 

Dr. Mary Fink in collaboration with other 
members of the Branch, Institute, and outside 
laboratories has continued and extended. her 
studies which are designed to evaluate exist- 
ing techniques and to devise new methods for 
the serological and immunological detection 
and characterization of viruses from ani- 
mal and human neoplasms. In particular, 
during the past year, these studies have been 
concerned with the techniques of immuno- 
fluorescence, micro-Ouchterlony (double diffu- 
sion in gels) and with specific hemagglutination 
of antigen coated tanned erythrocytes. In a lon- 
gitudinal study of the bone marrows from 26 
cases of acute human leukemia, 79% of the 
bone marrow specimens from patients in re- 



lapse showed positive immunofluorescence. 
Only 25 % of the bone marrow specimens from 
patients in remission showed positive immuno- 
fluorescence. In 6 of 8 human leukemia patients, 
followed through several cycles of relapse- 
remission, there was no evidence of the pres- 
ence of immunofluorescent cells during remis- 
sion. Dr. Fink and co-workers continued to test 
for immunofluorescence many different mate- 
rials received from investigators throughout 
this country and abroad. In regard to the 21 
different lines of human leukemia or lymphoma 
cells now maintained in tissue culture, the im- 
munofluorescent findings parallel the finding of 
herpes-like virus in these cultures. In collab- 
oration with Drs. Manaker and Dalton, several 
of these human lymphoma cell lines were ex- 
amined for the purpose of establishing the opti- 
mal time during tissue culture for demonstrat- 
ing immunofluorescence. These studies showed 
the fluorescence increases up to a maximum on 
the fourth or fifth day after fluid change. Vi- 
rus particles are also more numerous as de- 
tected by electron microscopy 4 or 5 days after 
fluid change. These and other studies designed 
to control the specificity of the immunofluores- 
cent reaction have been essentially confirmed 
in the laboratories of Drs. Yohn and Grace of 
the Roswell Park Memorial Institute and in lab- 
oratories of Dr. Zarafonetis at the University 
of Michign. A microdiffusion precipitation 
method using agar gels (Ouchterlony) was 
successfully adapted to the detection of several 
murine leukemia viruses. With the Rauscher 
strain, virus could be detected in the plasmas 
of viremic BALB/c mice as soon as 13 days 
post infection. Preliminary studies have indi- 
cated that the Rauscher and Friend viruses 
share at least 2 antigens but differ in at least 
1 and that the Moloney leukemia virus shares 
at least 1 antigen with the Rauscher virus. 
This technique can be used not only to detect 
murine leukemia viruses but also to quantitate 
the amount of virus present in various systems 
and to differentiate the various murine leu- 
kemia viruses. A third technique which has 
been developed and applied to the detection and 
assay of murine leukemia virueses and their 
antiserums is that of hemagglutination of anti- 
gen coated tanned erythrocytes. This tech- 



80 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



nique is not only useful as a quantitative 
measurement of antibody against murine leu- 
kemia viruses but also, by blocking the reac- 
tion with antigen, the technique becomes an 
exquisitely sensitive test for the presence and 
amount of virus in materials of unknown po- 
tency. As little as 0.2 micrograms per ml of 
antigen protein can be detected with this tech- 
nique. 

Each of the 3 above mentioned techniques 
are being intensively applied to the detection 
and quantitation of viruses and/or virus anti- 
gen presumed to be present in selected mate- 
rials obtained from human leukemia and lym- 
phoma patients. These in vitro monitoring 
techniques are of paramount importance to the 
human leukemia problem because it has not yet 
been possible to develop a sensitive laboratory 
animal system (including primates) which will 
support replication of and/or disease induction 
by candidate human leukemia viruses. It be- 
comes necessary, therefore, to employ in vitro 
serological techniques not only to monitor for 
the presence of virus in laboratory systems but 
also to conduct sero-epidemiological surveys 
for the presence or absence of experience with 
selected viruses in different human population 
groups. 

Dr. Robert Zeigel and staff continued their 
numerous collaborative activities with scien- 
tists of this Branch as well as those associated 
with other areas of NIH. In collaboration with 
Dr. Mary Fink, they obtained electron micro- 
scopic evidence for the presence of significant 
amounts of virus in mice 5 days following in- 
fection with a murine leukemia virus 
(Rauscher strain). This finding is important 
because it shows that large quantities of virus 
are present in animals shortly after infection 
and long before the infection process culmi- 
nates in frank disease. 

Dr. Zeigel's studies have also shown that 
tissue cultured cells which support active repli- 
cation of murine leukemia viruses are also ca- 
pable of phagocytosing virus particles produced 
and liberated by these same cells. This finding 
suggests a mechanism for a cyclic reinfection 
of cells and/or a means for the clearance and 
potential inactivation of extracellular infec- 
tious virus. Studies performed in collaboration 



with Dr. Gordon Theilen, a visiting investigator 
of NCI during fiscal year 1966, established the 
ultrastructure of avian reticuloendotheliosis vi- 
rus. Although this virus produces a leukosis 
like disease, it appears to be morphologically 
different from other viruses of the avian leu- 
kosis complex. 

During the past year Dr. Zeigel confirmed a 
extended his findings that type "A" virus par- 
ticles are associated with all tumors induced in 
conventional and germfree BALB/c mice with 
tumor viruses and/or with chemical carci- 
nogens. It has been shown that the endoplasmic 
reticulum of cells "infected" with type "A" par- 
ticles is substantially modified. This finding is 
of potential importance in view of the possibil- 
ities that "A" type particles may (a) serve as 
biological precursors for the eventual appear- 
ance of leukemogenic "C" type particles, (b) 
interfere with the replication of virus or the 
induction of disease by "C" type particles, and 
(c) may serve as a helper virus for the repli- 
cation of and/or diease induction by "C" 
type particles. 

Studies conducted in the laboratory of Dr. 
Due Nguyen, a visiting investigator, who 
joined the VLLB in August of 1965, are aimed 
at the application, to tumor viruses, of classical 
detection and bio-assay methods developed and 
used for studies of the necrotizing or nontu- 
morigenic viruses. In particular, he has at- 
tempted to device an in vitro method for the 
bio-assay of murine leukemia viruses based on 
the production of interferon or the detection 
of interference of necrotizing viruses by the 
noncytopathogenic murine leukemia viruses. 
Studies are well under way in his laboratory 
to apply the ferritin labeled antibody tech- 
nique of electron microscopy, used so success- 
fully with influenze virus, for the detection and 
localization of murine leukemia viruses in in- 
fected cells. Dr. Nguyen was successful in es- 
tablishing a line of embryonic rat kidney cells 
in tissue culture. These cells were infected with 
a murine leukemia virus (Rauscher strain) 
and appear to support the replication of more 
recoverable murine leukemia virus than any 
other cell line available for this purpose. 

The goal of studies conducted by Dr. Tim- 
othy O'Connor as principal investigator is to 



NATIONAL CANCER INSTITUTE 



81 



provide an integrated background of physical, 
chemical and immunological data on animal 
leukemia viruses toward a determi nation of the 
possible association of a virus or viruses with 
human leukemia and lymphoma. The predomi- 
nant antigenic activity of murine leukemia 
virus (Rauscher strain) purified by density 
gradient centrifugation was shown by 3 differ- 
ent immunological techniques to be associated 
with the virus particle. It was also shown that 
both the Rauscher and Friend strains of murine 
leukemia virus after extensive purification on 
density gradients still showed a significant re- 
action with antiserum prepared against nor- 
mal BALB/c mouse tissue antigens. This was 
not entirely unexpected since collaborative 
studies with Dr. Guy de The showed by elec- 
tron microscopic techniques that purified mu- 
rine leukemia viruses were coated with low 
amounts of host-derived enzymes. Experiments 
are in progress to determine whether these 
virus-associated, host-derived antigens arise by 
adsorption or from true structural integration 
into the viron. 

Treatment of murine leukemia viruses with a 
combination of ether and the non-ionic deter- 
gent, Tween 80, resulted in emulsions which 
could be separated into ether and aqueous 
phases. Density gradient centrifugation of the 
ether extract yielded low density materials 
which contained considerable amounts of viral- 
specific antigen. It was not possible to definitely 
determine the morphological characteristics of 
these components. Density gradient centrifu- 
gation of the aqueous phases yielded materials 
having a density of 1.24 to 1.27. These mate- 
rials, therefore, are much more dense than the 
intact virus and in addition did not react with 
viral-specific antisera or with antiserums pre- 
pared to BALB/c mouse antigens. Electron 
microscopically, 2 types of "subviral" particles 
were seen. The most common type was a pol- 
ygonal structure, the center of which appeared 
to be occupied with a fibrillar material of low 
electron density. These particles may represent 
the naked nucleoids of the mature virus par- 
ticle. The second type of subviral structure 
appeared as a rigid circular structure with an 
electron lucent core and in some instances ap- 
peared to have a helical structure composed of 



morphological subunits. These structures may 
represent the nucleoids of immature virus par- 
ticles. These studies are important because 
they may make it possible to compare the chem- 
ical and physical characteristics of intact and 
degraded murine leukemia viruses to similar 
characteristics of viruses isolated from human 
leukemia and lymphoma materials. Dr. O'Con- 
nor's studies have made it possible to apply 
density gradient centrifugation to the routine 
quality control of virus production. This has 
been done in his own laboratory and, with his 
monitoring collaboration, is now being rou- 
tinely conducted in commercial laboratories 
engaged in the production of large quantities 
of murine leukemia viruses under contract to 
the National Cancer Institute. This test is sim- 
ple and rapid and provides an excellent esti- 
mate of the relative homogeneity and physical 
titer of different batches of murine leukemia 
viruses produced in different laboratories at 
different times. 

Members of the Branch served as Project 
Officers on 9 contracts, the total funding level 
for which was approximately $3.7 million. The 
workscopes of these contracts include funda- 
mental and applied studies on animal and hu- 
man leukemias, research services, and the pro- 
duction of large quantities of highly purified 
concentrates of infective murine leukemia vi- 
ruses. One of these contracts deserves special 
mention because it provides a critical resource 
and unique technical competence to the re- 
search activities of the Branch as well as to 
the entire Special Virus-Leukemia Program. 
The objectives of this contract (with Bionetics 
Research Laboratories, Inc.) are: (a) to deter- 
mine whether newborn, mother-deprived pri- 
mates of various species are susceptible to the 
oncogenic and/or leukemogenic effects of known 
viruses and of candidate viruses recovered di- 
rectly from man, and (6) to test available 
means and develop new means to enhance the 
susceptibility of primates to virus replication 
and/or disease induction. Pursuant to these ob- 
jectives, the contractor has provided, largely 
from his own breeding colony, over 700 new- 
born viable primates suitable for inoculation. 
These animals have been inoculated with high 



82 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



priority materials received from over 50 differ- 
ent investigators from 40 different laboratories 
throughout this country and abroad. 

Members of the Branch presented, by invi- 
tation, over 40 lectures on their studies and on 
the studies of others at various meetings and 
to many different research groups in this 
country and abroad. Considerable time was de- 
voted to training, advising and collaborating 
with many different scientists from intramu- 
ral and extramural laboratories. In particular, 
Dr. Mary Fink and staff have trained investi- 
gators in the theory and use of the immuno- 
fluorescent technique reported by Drs. Fink 
and Malmgren for the detection and monitoring 
of an antigen which appears to be associated 
predominantly with the cells of human leuke- 
mia and lymphoma patients. Similarly, Dr. 
Timothy O'Connor has trained visiting investi- 
gators in his own laboratory as well as in lab- 
oratories throughout this country in the use of 
density gradient centrifugation and other bio- 
chemical and biophysical techniques for the 
purification and characterization of viruses 
and of subviral products. During the past year 
members of the Branch have dispensed liter 
quantities of different types of murini leuke- 
mia viruses, and advise on effective utilization 
of these viruses to over 120 different investi- 
gators. 

Drs. O'Connor, Fink, and Rauscher served 
on several different panels and committees 
among which were the Field Studies Contract 
Review Committee, the NCI Primate Study 
Group, Program Segment Working Groups of 
the Special Virus-Leukemia Program, and as 
members of the Civil Service Microbiology and 
Chemistry Panels. 

Special Virus-Leukemia Program 

During past year overall management of the 
Special Virus-Leukemia Program, which was 
planned and implemented in September of 1964 
through a special appropriation, was placed 
within the Office of the Chief, Viral Leukemia 
& Lymphoma Branch. The main objectives of 



this Program are: (1) to determine whether 
viruses comparable to those now known to be 
associated with avian and murine leukemia 
are etiological agents of human leukemia, and 
(2) to develop an effective vaccine or other 
means for the prevention and/or control of 
human leukemia and lymphoma if such etiolog- 
ical agents are found. The main assumption 
or working hypothesis on which the overall 
Program is based is that at least one virus 
is an indispensable element for the induction 
(directly or indirectly) of at least one kind 
of human leukemia (including lymphoma) and 
that the virus persists in the diseased indi- 
vidual. The overall Program was originally 
planned and modified during and following 
numerous discussions with key program lead- 
ers and research scientists of NCI as well as 
with university and industrial personnel expert 
in the general areas of virology, oncology, 
chemotherapy, etc. From the research stand- 
point the program is divided into 4 major 
areas of effort: Human Leukemia Etiology and 
Prevention, Special Animal Leukemia Ecology 
Studies, Biohazards Control and Containment, 
and Human Leukemia Therapy. Operationally 
the Program has been divided into seven pro- 
gram segments: Developmental Research, Test- 
ing and Monitoring, Resources and Logistics, 
Epidemiology, Special Animal Leukemia Ecol- 
ogy Studies, Human Leukemia Therapy, and 
Biohazards Control and Containment. These 
working groups, which with the exception of 
the Human Leukemia Therapy Segment, are 
chaired by senior scientists within the Etiology 
Area who are responsible for the development 
of research programs in their respective seg- 
ments within the context of the overall plan. 
Approximately 70 of 180 projects, which cur- 
rently make up the program plan, are being 
conducted by investigators in governmental 
laboratories and clinical facilities, in universi- 
ties, in nonprofit laboratories, and in commer- 
cial facilities. Selection and implementation of 
all projects are done on the basis of both sci- 
entific excellence and high priority relevance 
in terms of the integrated program. 



NATIONAL HEART INSTITUTE 



CARDIOLOGY BRANCH 

The fundamental objective of the research 
program of the Cardiology Branch, NHI is to 
provide an increased understanding - of the de- 
rangements in cardiac function which occurs 
in various forms of heart disease. In the pur- 
suit of this long-range objective it has become 
evident that the mechanics and energetics of 
the normal contractile process are poorly un- 
derstood and that gross clinical and hemody- 
namic studies on diseased hearts provide only 
superficial descriptions of disease processes. 
Accordingly, efforts have been underway for 
several years to analyze the behavior of the 
normal and diseased heart as a pump. More 
recently, these approaches have been broadened 
and cardiac performance is now also being 
analyzed from the point of view of the heart 
as a muscle. As in the past, investigations are 
simultaneously being carried out on isolated 
heart muscle, intact canine hearts, diseased 
canine hearts, as well as on patients with nor- 
mal and diseased cardiovascular systems. 

Mechanics and Energetics of Myocardial 
Contraction: Studies on Isolated Heart Muscle 

The active state of muscle has been defined 
as a mechanical measure of those processes in 
the contractile elements which generate force 
and are responsible for shortening. In a study 
designed to determine the manner in which 
the active state in heart muscle is established 
and dissipated it was found that: (1) in con- 
trast to skeletal muscle, the onset of maximum 
active state in heart muscle is delayed, de- 
veloping 100 to 150 msec, after the first evi- 
dence of active state; (2) the maximum active 
state is maintained for about 100 msec, and 
does not decline until just prior to the develop- 
ment of maximum active tension; and (3) 



inotropic interventions which alter the con- 
tractile state of the muscle, such as changing 
frequency of contractions, or norepinephrine, 
accelerate the onset, increase the intensity, and 
hasten the decline of the active state. 

There has long been controversy as to 
whether stimuli which exert a positive ino- 
tropic effect on the myocardium also induce 
a change in the true compliance of the heart 
muscle. Since these inotropic interventions are 
generally associated with substantial incre- 
ments in the force of contraction, the possi- 
bility was considered that the changes in com- 
pliance, were they to occur, might be mediated 
through increases in systolic force per se 
rather than through a direct effect of the ino- 
tropic intervention on the contractile system 
of the muscle. However, if changes in diastolic 
compliance of heart muscle are indeed due to 
alterations in the contractile system, they 
should be apparent whether a muscle is con- 
tracting under isotonic or isometric conditions. 
On the other hand, should changes in systolic 
force alone explain these findings, then no 
change in compliance should be observed under 
isotonic conditions, but should occur when- 
ever systolic force is increased, regardless of 
whether or not an inotropic influence is intro- 
duced. In a study on isolated papillary muscles 
it was observed that postextrasystolic poten- 
tiation, norepinephrine or calcium administra- 
tion produced no change in diastolic compli- 
ance in the isotonically contracting muscle or 
in the afterloaded muscle in which increased 
shortening occurred with no change in force. 
However, isometric contraction consistently in- 
duced a small fall in diastolic tension when 
the force of contraction rose. It was concluded 
that inotropic interventions per se do not in- 
duce a true change in myocardial compliance. 
However, diastolic compliance may increase 



83 



84 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



when the force of contraction rises. These 
findings provide evidence for a series viscous 
component in heart muscle which is altered 
when changes in contractile force occur. 

A study has also been initiated to compare 
the magnitude of stress relaxation that occurs 
in series viscous elements of isolated cardiac 
muscle with that occurring in another com- 
ponent which had not been defined previously 
and which has been termed the "parallel vis- 
cous component." This parallel viscous com- 
ponent appears to be several times as extensi- 
ble as the series viscous component. It also 
appears that the interplay between effects due 
to these two viscous elements and their op- 
posite effects on the resting length of the con- 
tractile element may serve to explain many 
of the phenomena associated with homeometric 
autoregulation of the heart. 

The series elastic is one of the fundamental 
components of heart muscle and its properties 
must be characterized in order to allow an 
understanding of the behavior of the con- 
tractile elements. Accordingly, the series elas- 
ticity of cat papillary muscle was determined 
by a variety of techniques and revealed an 
extension of 5 to 10% of muscle length at a 
developed force of 10 gm. Inotropic interven- 
tions did not alter the series elastic compon- 
ent and calculation of contractile element ve- 
locity revealed that in auxotonic contractions 
contractile element velocity exhibits a secon- 
dary rise during the transition from the 
isometric to the isotonic phase. Data were also 
obtained to support the position that a portion 
of the series elastic is in series with both the 
contractile elements and the parallel elastic 
elements. 

Although ultrastructural and biochemical 
similarities are known to exist in the contrac- 
tile systems of cardiac and skeletal muscle 
fundamental differences in their force gener- 
ating processes have been postulated. This 
view is based on a ten-fold disparity in force 
generating capacity between the two types of 
muscle, which has previously been reported. 
However, an accurate comparison of the force 
generating capacity of cardiac and skeletal 
muscle depends upon establishing a maximum 
contractile state of cardiac muscle and utiliz- 



ing electronmicroscopic analyses to correct for 
its greater proportion of non-contractile ele- 
ments such as mitochondria, nuclei and inter- 
filamentous spaces. When this is applied to 
the cat papillary muscle preparation, the max- 
imum contractile force of cardiac muscle 
achieved during paired electrical stimulation 
was found to be comparable to that reported 
for skeletal muscle, suggesting that the force 
generating mechanisms are basically similar 
quantitatively. 

The mitral valve is generally thought to con- 
sist of elastic and fibrous tissue and its motion 
has been considered entirely passive, the re- 
sults of changes in ventricular and atrial pres- 
sures. However, this view was re-evaluated by 
assessing the mechanical properties of the mi- 
tral valve in vitro. The anterior leaflet of the 
mitral valve was placed in a myograph. With 
stimulation, it was found that the valve con- 
tracts actively and as with other contractile 
tissues, the force of contraction was found 
to be a direct function of length. Electronmi- 
croscopic study of the mitral valve has con- 
firmed the presence of heart muscle under- 
neath the valve. These studies have demon- 
strated the need for a total re-evaluation of 
the control of mitral valve motion both in nor- 
mal and pathological states. 

It is well known that stimulation of cardiac 
sympathetic nerves and the administration of 
norepinephrine (NE) increase the force of car- 
diac contraction. Thus, the sympathetic nervous 
system provides a mechanism for augmenting 
basal cardiac contractility, but it has not been 
known whether or not the cardiac stores of 
NE are necessary to establish normal baseline 
contractility and to maintain the potential for 
increasing contractility in response to posi- 
tive inotropic interventions other than sympa- 
thetic stimulation. This question is of particular 
interest in view of the depletion of myocardial 
NE stores associated with congestive heart 
failure. Accordingly, in order to assess the role 
played by endogenous norepinephrine (NE) 
stores in the intrinsic contractile state of car- 
diac muscle, the right ventricular papillary 
muscles from normal cats and cats with car- 
diac NE depletion produced by chronic car- 
diac denervation or reserpine pretreatment 



NATIONAL HEART INSTITUTE 



85 



were studied. Length-tension curves, force- 
velocity relations, the effects of alterations in 
frequency of contraction and the response to 
sustained postextrasystolic potentiation were 
determined. In addition, the electrical excita- 
bility and absolute refractory periods were 
measured. All of these properties were found 
to be normal in both groups of NE depleted 
muscles. It is concluded that cardiac stores of 
NE are not fundamental for maintaining the 
intrinsic contractile state of the myocardium, 
its absolute refractory period or electrical ex- 
citability. Further, release of endogenous NE 
from cardiac muscle does not appear to play 
an essential role in the mediation of the posi- 
tive inotropic effects of increasing frequency 
of contraction or of sustained postextrasystolic 
potentiation. 

Major interest has been directed to the ef- 
fects of sympathetic nervous system activity 
on cardiac contractility, but far less is known 
about the action of the parasympathetic ner- 
vous system. Acetylcholine (ACh), the para- 
sympathetic neurotransmitter, is said to exert 
a positive inotropic effect on the mammalian 
ventricle, an effect which has been attributed 
to the release of stored NE. In order to test 
this hypothesis, the effect of ACh on normal 
cat papillary muscles was compared to the re- 
sponse of muscles from hearts depleted of NE 
by chronic cardiac denervation and reserpine 
pretreatment. In the absence of NE the posi- 
tive response produced by ACh is unchanged 
from that observed in normal muscles. By ex- 
amining the response to ACh over a wide con- 
centration range and comparing it to that in 
isolated atrial tissue in the presence and ab- 
sence of atropine, the following hypothesis 
was developed to account for an otherwise be- 
wildering array of apparently contradictory 
data on parasympathetic control of the heart: 
There appear to be two types of receptor sites 
in the heart responsive to acetylcholine — Type 
I, intimately related to vagal nerve endings 
and whose stimulation is associated with a 
negative inotropic effect and Type II, unre- 
lated to the vagus nerve and whose stimula- 
tion results in a positive inotropic effect. This 
study thus emphasizes the need for a reexami- 
nation of the role of the parasympathetic ner- 



vous system in the control of myocardial con- 
tractility. 

The direct influence of the thyroid state on 
cardiac muscle has not been denned. While 
profound cardiovascular alterations are known 
to occur with hyper- and hypothyroidism, it 
is not clear from studies in the intact organism 
whether these result from fundamental changes 
in the heart muscle itself or merely reflect the 
heart's response to the systemic effects pro- 
duced by these endocrine disorders. The papil- 
lary muscles isolated from cats rendered hy- 
perthyroid and hypothyroid provides a prepa- 
ration in which it is possible to exclude as 
much as possible the role that neurohumoral 
and metabolic factors might play in indirectly 
altering the contractility. 

The level of thyroid state was found to have 
a profound influence on the intrinsic con- 
tractile state of isolated papillary muscles. 
Those parameters related to the speed of con- 
traction, i.e. the V m «, the time to peak ten- 
sion, the rate of tension development, and 
latency were particularly affected, in a manner 
indicating that the basic processes controlling 
the rate of force generation are accelerated 
by increased levels and slowed by decreased 
levels of thyroid hormone. These effects were 
found to be independent of temperature, fre- 
quency of contraction, and endogenous NE 
stores. The level of thyroid hormone was found 
to have less effect on contractile force than on 
the speed of its development, but the positive 
inotropic response to paired electrical stimula- 
tion, NE, and strophanthidin are dependent on 
the thyroid state. Thus, it appears that thy- 
roid hormone may directly affect the con- 
tractile state of heart muscle and condition 
the responsiveness of the heart to other agents 
that act upon it. 

In order to define the chemical energetics of 
cardiac muscle, the utilization of ATP and crea- 
tine phosphate (CP) in the papillary muscle 
of cat heart was measured. To accomplish this, 
cat right ventricular papillary muscles were 
poisoned with iodoacetic acid and nitrogen to 
prevent further production of ATP and CP. 
The basal consumption of high energy phos- 
phate was determined in resting muscles with- 



86 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



out tension by freezing muscles at various times 
after poisoning and assaying these muscles 
for CP and ATP. It was found that the 
utilization of high energy phosphates could be 
accounted for entirely by the disappearance 
of CP, indicating that creatine phosphokinase 
was intact. 

The basal rate of CP consumption was 0.71 
/xmoles/g/min. Increasing resting tension 
(or muscle length) was found to increase basal 
CP consumption to a linear fashion over the 
physiologic range of muscle lengths. In addi- 
tion, CP utilization was determined in 49 iso- 
metrically contracting muscles stimulated to 
contract 10 to 50 times at the top of their 
length-tension curves. The utilization of CP by 
these muscles could be accounted for by a pre- 
diction equation with terms for the number of 
activations and the calculated contractile ele- 
ment work. The mechanochemical efficiency of 
cardiac muscle, defined as the work done di- 
vided by the energy cost of work plus activa- 
tion, averaged 39% in these studies. This is 
similar to the efficiency of skeletal muscle pre- 
viously determined by other investigators. 

The in vivo steady state levels of myocardial 
high energy phosphate stores (adenosine tri- 
phosphate-ATP and creatine phosphate-CP) 
may indicate the relative balance of energy 
production and utilization in the heart. Tech- 
niques developed in this laboratory have made 
it possible to determine in vivo stores of ATP 
and CP in rapidly frozen myocardial biopsies. 
Three groups of cats were studied in addition 
to normal controls: (1) cats with right ven- 
tricular hyertrophy and failure; (2) cats 
made hyperthyroid; and (3) cats made myxe- 
dematous. Right ventricular stores of ATP 
were not found to be significantly different in 
these groups. Right ventricular stores of CP 
were found to be depressed in hyperthyroid 
and pulmonary artery constricted cats. Right 
ventricular creatine stores were depressed in 
all three states. These findings indicate that 
the contractile state of the heart is not de- 
pendent on its high energy phosphate stores. 
In hyperthyroid cats, the absolute depression 
of CP stores may be related to a depression of 
total creatine stores. In pulmonary artery con- 



stricted cats, the low CP/total creatine ratio 
may indicate a relatively higher demand/ 
production ratio for chemical energy. 

Application of Myocardial Mechanics to the 
Intact Heart 

In order to increase understanding of the 
gross architecture and the ultrastructure of 
the left ventricle during various phases of the 
cardiac cycle, a technique was developed for 
the rapid fixation of the canine left ventricle 
during various phases of the cardiac cycle. A 
Gregg cannula was placed in the left main 
coronary artery, and at a selected time during 
diastole or systole, the fixing agent glutaral- 
dehyde, preceded by a bolus containing potas- 
sium chloride and acetylcholine, was injected 
with a power syringe. Silastic casts of the 
left ventricles were then prepared, and ven- 
tricular volumes measured directly. Portions of 
the myocardial wall were prepared for elec- 
tron microscopy, sectioned and analyzed for 
sarcomere lengths. Sarcomeres of the ven- 
tricular wall have averaged 2.10 /x during 
diastole and 1.84 n during systole. In the 
acutely dilated heart, sarcomeres of more than 
2.25 jx have been observed with the forma- 
tion of H zones. Analyses of the gross dimen- 
sions of the fixed ventricle and of the silastic 
cast of its chamber have also been undertaken. 
Data have been obtained on ventricular wall 
thickness at the apex and at the waist of the 
left ventricle during systole and diastole, and 
on the base to apex and base to outflow tact 
dimensions. 

The relations between tension and the ve- 
locity of shortening in the intact left ventricle 
of the dog were examined in a manner ana- 
logous to that employed in isolated muscle, i.e. 
by serial, reproducible variations in the after- 
load alone, from a constant end-diastolic vol- 
ume. Sudden increases or decreases in the aortic 
pressure during diastole were produced, and 
ejection rate was measured with an electro- 
magnetic flowmeter; left ventricular wall ten- 
sion and the shortening velocities of the myo- 
cardial fibers and the contractile elements were 
then calculated. By analyzing isovolume points 
early in ejection, effects resulting from two 



NATIONAL HEART INSTITUTE 



87 



other determinants of shortening velocity, i.e. 
duration of the active state and the instan- 
taneous muscle length, were minimized. Shifts 
in the basic force-velocity relation with altera- 
tions in V m ax, obtained by extrapolation, and 
maximum tension were clearly demonstrated. 
Norepinephrine and paired electrical stimula- 
tion caused a shift in this relation to the right, 
with increases in velocity at any tension. Sim- 
ilar shifts were also apparent in curves relating 
tension to velocity, calculated during single 
isovolumic contractions. It was suggested that 
determination of these relations expands tra- 
ditional definitions of ventricular performance, 
and that estimation of changes in maximum 
velocity as well as maximum strength relative 
to muscle length provides direct information 
concerning alterations in the contractile state 
of the intact heart. 

The contractile state of the intact canine 
left ventricle was then studied by analyzing 
the instantaneous relations between force and 
contractile element velocity (V CE ) during the 
course of single isovolumic beats, and the sensi- 
tivity of this relation was compared to that of 
the ventricular function curve by exerting 
small inotropic influences. In 7 dogs, norepine- 
phrine always shifted the isovolumic force- 
velocity (FV) curve, increases occurring both 
in maximum V CE and maximum tension (Po); 
the ventricular function curve was unchanged 
in 4 of the 7 dogs. Moderate hypothermia (avg. 
30.8° C) in 4 dogs increased P p , with no 
change or a fall in maximum V CE ; the ven- 
tricular function curve was shifted upward 
and to the left in 2 of these dogs and un- 
changed in 2. In 4 dogs moderate increases in 
heart rate (avg. 32 beats/min) increased the 
maximum V CE , with little change in P ; no 
shifts in the ventricular function curve oc- 
curred. Thus, the isovolumic FV curve was 
more sensitive than the ventricular function 
curve in detecting changes in myocardial con- 
tractile state, and provided more complete 
definition of alterations in left ventricular per- 
formance by allowing separation of effects due 
to changes in shortening velocity from those 
due to alterations in the strength of myocar- 
dial contraction. 



The effects of increasing heart rate on the 
relationship between contractile element ve- 
locity and myocardial wall tension were de- 
termined from single isovolumic contractions 
in 9 canine right heart bypass preparations. 
Increases in heart rate ranging from 10 to 70 
beats per minute were produced by crushing 
the sino-atrial node and stimulating the right 
atrium. An increase in heart rate always pro- 
duced an increase in maximum calculated con- 
tractile element velocity and in other variables 
reflecting the speed of contraction, such as 
peak aortic flow rate, the peak first derivative 
of the left ventricular pressure pulse, peak con- 
tractile element power and the time to peak 
isovolumic tension. Maximum isovolumic ten- 
sion usually increased. It is concluded that in 
the intact canine left ventricle, increasing the 
frequency of contraction always increases the 
maximum contractile element velocity and 
maximum isovolumic force of contraction. 

The effect on contractile force of changing 
heart rate had not been determined directly 
in man, and accordingly a study was under- 
taken to provide definitive information con- 
cerning this relationship. At the time of cor- 
rective cardiac surgery a Walton-Brodie strain 
gauge arch was sutured to the right ventricle 
of 8 patients. In man, a "velocity staircase" 
i.e. an increase in the rate of tension develop- 
ment rather than a "force staircase" i.e. an 
increase in the peak tension development, oc- 
curs with changes in heart rate. This mecha- 
nism apparently permits the human ventricle 
to maintain its force of contraction and to pre- 
serve the duration of diastolic filling with in- 
creases in rate. 

It has been suggested that serotonin, known 
to act on vascular smooth muscle and known 
to be present in the heart, may play a role in 
controlling cardiac contractility. The direct ef- 
fects of serotonin on cardiac muscle have been 
examined in the isolated cat papillary muscle 
and in the intact dog heart, utilizing the right 
heart bypass isovolumic preparation described 
above. Preliminary results indicate that sero- 
tonin produces an increase in isometric ten- 
sion in the isolated muscle and a shift in the 
isovolumic force-velocity relationship to the 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



right, both observations reflecting a positive 
inotropic effect. The physiologic significance of 
these findings is currently under investigation. 

An attempt has been made to determine 
whether the technique of sudden occlusion of 
left ventricular outflow could be applied to the 
study of force-velocity-length relationships in 
the intact unanesthetized dog. In addition, ef- 
forts have also been directed to deriving, for 
the first time, normalized values for the force- 
velocity-length relations in a group of normal 
intact dogs and to use these as a basis for com- 
parison with the relations obtained in dogs 
subjected to various chronic interventions. 

Several weeks before the experimental proce- 
dure a thoracotomy is performed. The peri- 
cardium is sutured to the left chest wall to 
facilitate later percutaneous left ventricular 
puncture. Isovolumic left ventricular contrac- 
tions are produced by balloon occlusion of the 
ascending aorta during diastole. Analysis of 
left ventricular pressure and the rate of de- 
velopment of pressure in isovolumic beats has 
been found to give meaningful force-velocity- 
length relationships in the lightly sedated, 
closed chest dog. Blood volume loading, by in- 
creasing cardiac filling and end-diastolic left 
ventricular fiber length, shifts the force-ve- 
locity curve to the right with an increase in 
maximum isovolumic tension development but 
produces no change in maximum velocity of 
contractile element shortening. Positive ino- 
tropic influences (digitalis glycosides, catecho- 
lamines, and paired electrical stimulation) 
produce changes in the force-velocity-length 
relationships similar to those seen in isolated 
cardiac muscle. Beta-adrenergic blockade in the 
intact dog depresses the left ventricular force- 
velocity curve, both in the presence and ab- 
sence of digitalis glycosides. In addition, the 
relationships between left ventricular tension 
and shortening in ejecting contractions, and 
maximum tension development in isovolumic 
beats promises to provide further insight into 
the mechanics of left ventricular function in 
the normal intact animal. 

As indicated above, it has been shown in 
studies in the intact dog heart that the rela- 
tion between instantaneous myocardial wall 



tension and the velocity of fiber shortening 
during systole provides a sensitive means of 
examining the contractile state of the left ven- 
tricle (LV). Previously, it has not been pos- 
sible to quantify this important relation in the 
human LV. A study was therefore undertaken 
to determine the course of tension development 
during systole and the velocity of circumferen- 
tial shortening of the human LV and to ex- 
amine the instantaneous tension-velocity rela- 
tion in patients with and without LV dys- 
function. 

In 15 patients, LV pressure was measured 
continuously with a cathetertip transducer, 
while radiographic contrast material was in- 
jected into the left atrium. The true radius 
(r) of the minor LV circumference (circ.) 
was then determined at 170 msec, intervals 
from cineangiograms, and this measurement 
correlated with instantaneous LV pressure 
(Pr), the tracing of which was recorded di- 
rectly on the cine film. The velocity of circum- 
ferential shortening (V CF ) was calculated as 
2 n dr/dt. Wall tension in the corresponding 
slice of muscle was computed as: 

Prxr 



Wall thickness 



In patients without LV disease, the maxi- 
mum V CF averaged 2.05 circ./sec.; the cor- 
responding wall tensions ranged from 374 to 
384 gm./cm 2 . In the patients who exhibited 
other hemodynamic evidence of LV disease, 
the maximum VCF's were consistently lower 
and averaged only 0.95 circ./sec, at levels of 
wall tension comparable to those observed in 
patients without evidence of LV dysfunction. 

These measurements provide the first de- 
scription of the tension-velocity relation in the 
intact human LV. In addition, the normalized 
measurements of VCF allow comparisons of the 
contractile state of the LV in patients with and 
without LV dysfunction. These preliminary re- 
sults indicate that the contractile state of the 
LV, as determined from the basic myocardial 
tension-velocity relation, can be assessed by 
the technique employed, and clearly serves to 
distinguish normal from abnormal LV func- 
tion. 



NATIONAL HEART INSTITUTE 



89 



Contractile Properties of the Failing Heart 

Studies on Isolated Heart Muscle and Sub- 
cellular Fractions 

It has long been appreciated that a quanti- 
tative definition of the contractile state of car- 
diac muscle obtained from hypertrophied and 
failing hearts is needed. The isolated cat papil- 
lary muscle preparation allows quantification 
of intrinsic cardiac contractility per unit of 
muscle, as well as determination of the re- 
sponses of heart muscle to inotropic interven- 
tions. The contractile properties of normal 
papillary muscles can be compared with cor- 
responding muscles from abnormal hearts. Ac- 
cordingly, techniques were developed for the 
production of right ventricular hypertrophy 
and right heart failure in cats by graded con- 
striction of the main pulmonary artery. 

Doubling of right ventricular weight occurs 
within several weeks after constriction. When 
the degree of pulmonary artery constriction is 
less severe, ventricular hypertrophy occurs 
with right ventricular systolic hypertension 
but without evidence of heart failure. In ani- 
mals in which the pulmonary artery is con- 
stricted more severely, hypertrophy is 
accompanied by heart failure with visceral 
congestion, ascites, pleural effusion, elevated 
right ventricular end-diastolic pressure and de- 
creased cardiac output. 

The muscles from cats with cardiac hyper- 
trophy and heart failure demonstrated pro- 
found depressions of the maximum isotonic 
velocity of shortening as well as of the max- 
imum isometric tension. It is concluded that 
congestive heart failure is associated with pro- 
found abnormalities in the contractile state 
per unit of heart muscle, with reduction of 
both the functional level of contractile state 
and its ceiling or maximum. These abnormali- 
ties do not appear to be dependent on an al- 
teration of the normal relation of sarcomere 
lengths to length-tension dynamics. Further it 
is tentatively proposed that ventricular hyper- 
trophy alone, in the absence of demonstrable 
heart failure, is associated with depression of 
contractile state per unit of cardiac muscle, 
although the absolute increase in total muscle 



mass may be sufficient to maintain cardiac 
compensation. 

It has been the general impression that the 
hypertrophied ventricle is less distensible than 
normal. This might be due either to an increase 
in the total muscle mass or to the decreased 
compliance of individual fibers. Accordingly the 
effect of hypertrophy on the resting length- 
tension relationship of isolated segments of 
normal and hypertrophied rat ventricles was 
determined. It was observed that although the 
hypertrophied ventricles exceeded the normal 
ventricles by an average of 20% in weight, 
there was no evidence that the hypertrophied 
myocardium is less distensible than normal, 
if proper corrections for differences in weight, 
length, and cross-sectional area were made. 

Collagen comprises approximately 4 percent 
of the dry weight of heart muscle and may con- 
tribute to the diastolic compliance of the ven- 
tricle. The few investigations of the connective 
tissue content in cardiac hypertrophy have not 
revealed a change in the collagen to muscle 
ratio in hypertrophied ventricles. However, 
only the central part of the ventricular wall, 
after removal of the epicardial and endocardial 
surfaces, has been studied. Since the surfaces 
also contribute to the hemodynamic charac- 
teristics of the entire ventricular wall it 
seemed important to determine if any area of 
the hypertrophied ventricle had altered 
amounts of collagen. Accordingly, tissue col- 
lagen content was determined in normal cat 
ventricles and in those with right ventricular 
hypertrophy due to banding of the main pul- 
monary artery. Each ventricle was split into 
epicardial and endocardial halves for determi- 
nation of collagen content and the ultrastruc- 
ture of these muscles was also examined. The 
hypertrophied right ventricles had markedly 
and significantly increased quantities of col- 
lagen per unit of muscle, while the left ventri- 
cle from the same hearts had normal collagen 
values. The epicardial half of the right ven- 
tricles had the highest collagen content but the 
content was also increased in the endocardial 
half. Large bundles of connective tissue were 
evident in the ultrastructural studies of the 
right ventricles. 



90 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



In vitro function of mitochondria from fail- 
ing hearts has been variously reported as nor- 
mal and abnormal. A re-evaluation of the ques- 
tion of mitochondrial integrity in heart failure 
has become necessary because of: (1) the re- 
cent development of polarographic techniques 
offering more accurate data than manometric 
techniques; and (2) the ability to determine 
precisely the physiologic function of failing 
heart muscle. Chronic heart failure was in- 
duced in cats by tight constriction of the pul- 
monary artery and in guinea pigs by tight 
constriction of the aorta. The presence of heart 
failure in the cats was identified by: (1) in 
vivo cardiac catheterization indicating high 
end-diastolic pressures; (2) pathologic find- 
ings such as ascites and massive right ven- 
tricular (RV) hypertrophy; (3) depressed 
mechanical performance of the isolated RV 
papillary muscle studied in vitro. After isola- 
tion of mitochondria from the right ventricle 
(cats) and left ventricle (guinea pigs), polaro- 
graphic determinations of oxygen consumption 
(Q0 2 ), respiratory control ratio (RC), and 
the ratio of phosphate esterified with ADP to 
the oxygen consumed (P/O ratio) were car- 
ried out. Determinations were performed at 
26° C and 37° C with both pyruvate/malate 
and glutamate substrates. Initial studies have 
indicated no abnormalities of Q0 2 , RC or 
P/O ratio in mitochondria isolated from 
hearts in experimental heart failure. 

The possibility that there may be a bioen- 
ergetic defect in the myocardium involving an 
impairment of oxidative phosphorylation in 
heart failure in man was examined in myo- 
cardial tissue obtained from patients at the 
time of cardiac operations. Mitochondria were 
isolated from papillary muscles removed from 
the left ventricle during replacement of the 
mitral valve in 11 patients with left ven- 
tricular failure. Measurement of oxidative 
phosphorylation with pyruvate/malate as sub- 
strate gave P/O ratios averaging 2.8. Res- 
piratory control ratios, determined both mano- 
metrically and polarographically averaged 6.6 
with pyruvate/malate and 5.9 with alpha- 
ketoglutarate. Endogenous ATPase activity av- 
eraged 0.14 /xmoles P liberated per minute per 
mg mitochondrial N; it averaged 0.36 with 



Mg ++ , 1.79 with 2,4-dinitrophenol, and was 
completely inhibited by oligomycin. These 
values are comparable to those reported for 
normal mitochondria obtained from experi- 
mental animals. 

Examination of myocardial tissue by elec- 
tronmicroscopy revealed no apparent abnor- 
mality of mitochondrial size or crystal pattern. 
Creatine phosphate was determined in rapidly 
frozen ventricular biopsies from 15 patients 
with heart failure undergoing valve replace- 
ment. The mean tissue concentration was 4.0 
xmoles per g compared to 4.1/mioles per g in 
similar biopsies from 5 patients without heart 
failure. These biochemical studies indicate that 
electron transport and coupled phosphorylation 
appear to be normal in mitochondria isolated 
from failing human hearts and that there is 
no consistent reduction of the myocardial store 
of high energy phosphate. It is concluded that 
the formation of chemical energy is not im- 
paired in the failing heart, and it is suggested 
that the biochemical abnormality responsible 
for defective myocardial function involves 
utilization of energy in the contractile process. 

Studies on the Intact Dog Heart 

There is general agreement that both the 
high energy phosphate stores and the mechan- 
ical performance of the myocardium are de- 
pressed during severe hyposia. To determine 
whether a casual connection exists between 
these two phenomena, 18 dogs were respired 
with 6% 2 , 94% N 2 after sympathetic block- 
ade (hexamethonium and propranolol). Serial 
left ventricular (LV) biopsies were obtained 
as myocardial failure occurred, and the con- 
centrations of adenosine triphosphate (ATP) 
and creatine phosphate (CP) were measured. 
A transient increase in left ventricular stroke 
work during initial hypoxia was followed by 
a progressive rise in left ventricular end-dia- 
stolic pressure and fall in left ventricular 
stroke work. There was no change in mean ATP, 
even with severe heart failure. With early 
failure a significant fall in CP occurred in 10 
of 18 dogs, and with late failure CP was always 
depressed. Since ATP, the final energy source 
for contraction, was unaffected in all dogs and 



NATIONAL HEART INSTITUTE 



91 



CP, the secondary energy store, was not de- 
pressed when failure first developed in some 
animals, it is concluded that hypoxic depres- 
sion of myocardial function is not initiated by 
a depression of the total myocardial high en- 
ergy phosphate stores. 

The levels of high energy phosphate stores 
and the status of anaerobic metabolism at the 
onset of ischemic heart failure induced by re- 
stricting flow to the left coronary artery were 
also studied. Left ventricular biopsies were ob- 
tained before ischemia and shortly after the 
onset of ischemic heart failure for measure- 
ment of concentrations of ATP and CP. Sam- 
ples of blood were drawn from the aorta and 
coronary sinus for determination of lactate 
and pyruvate concentrations. At the time of 
failure, arteriovenous lactate differences de- 
creased from an average of 1.29 to 0.60 mM 
and "excess lactate" increased by an average 
of 1.74 mM. There was no change in mean 
ATP (control, 6.45 vs. failure, 6.49 ^moles/g). 
Mean CP concentrations fell significantly 
(12.84 to 8.00 ^moles/g) but concentrations 
of CP were within 2 SD's of paired control 
samples in 5 of 19 observations. It is con- 
cluded that heart failure may be induced in 
the presence of relatively minor degrees of 
anaerobic metabolism, and that the total high 
energy phosphate stores are not necessarily 
compromised at this time. 

Depressions of cardiac norepinephrine stores 
have been described in this laboratory in 
chronic heart failure in man and in experi- 
mental heart failure in the dog, cat and guinea 
pig. Disturbances have been noted in both the 
uptake and storage of exogenous norepine- 
phrine, although the relative rate of norepine- 
phrine turnover is normal. These findings have 
suggested that there may be an absolute loss 
of sympathetic nerve endings in the heart in 
chronic heart failure. Tyrosine hydroxylase is 
the rate limiting enzyme in the synthesis of 
norepinephrine. It was thought, therefore, that 
the determination of cardiac tyrosine hydrox- 
ylase activity might provide insight into 
the mechanism of the depletion of nore- 
pinephrine stores in heart failure. Initial 
studies of myocardial tissue extracts have dem- 
onstrated the possibility of assaying tyrosine 



hydroxylase activity in canine right ventricle 
and have suggested that the activity of this 
enzyme is low in chronic right ventricular 
failure in dogs. 

As outlined in an earlier section of this re- 
port, other studies in this laboratory have 
shown that analysis of the tension-velocity re- 
lation throughout a single isovolumic beat pro- 
vides a sensitive index of the contractile state 
of the intact left ventricle of the dog. Accord- 
ingly, the effects of acute experimental heart 
failure on tension-velocity relations during iso- 
volumic contractions and auxotonic contrac- 
tions were studied. Cardiac failure was pro- 
duced by infusion of large doses of the beta- 
adrenergic blocking agent pronethalol, or by 
infusion of barbiturates. The right heart by- 
pass preparation was used to control cardiac 
output. Blood flow was measured with 
an electromagnetic blood flow transducer 
positioned about the aortic root. When 
veutricular end-diastolic volume was main- 
tained constant, the tension-velocity re- 
lation during isovolumic contraction was al- 
ways shifted markedly downward and 
to the left during acute failure of the left 
ventricle, with marked reductions both of max- 
imum measured contractile element velocity, 
the extrapolation to V m «, and the maximum 
isovolumic tension (P ). Acute failure also 
produced a decreased extent of fiber shorten- 
ing and an increase in the ratio of the ten- 
sion actually developed (P), to P . In some 
experiments, stroke volume was held constant 
so that end-diastolic pressure and volume were 
increased during acute cardiac failure. Al- 
though, under these circumstances, maximum 
measured velocity and V max are depressed, 
P„ remains unchanged or more often is ac- 
tually increased during acute failure. Further- 
more, it appears that while total contractile 
element work may not be greatly affected, the 
ratio of internal contractile element work to 
total work may be increased substantially. 

A marked augmentation of myocardial con- 
tractile force regularly accompanies paired 
electrical stimulation, a technique in which two 
stimuli are repetitively delivered to the ventri- 
cle with such timing that the second stimulus 
of each pair is introduced at the termination of 



92 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the preceding refractory period. Profound pos- 
itive inotropic effects have been demonstrated 
in isolated heart muscle, in a variety of animal 
preparations and in conscious human subjects. 
However, the potential clinical usefulness of 
this technique has been sharply restricted by 
the finding that this augmentation of ventricu- 
lar contractility is not always translated into 
an elevation of cardiac output. Accordingly, an 
investigation was carried out in order to iden- 
tify circumstances in which the improvement 
of myocardial contractility produced by paired 
electrical stimulation would result in an eleva- 
tion of the cardiac output. 

The effects of paired stimulation were stud- 
ied in intact anesthetized dogs before and after 
barbiturate induced depression of myocardial 
performance. In the non-depressed state, paired 
electrical stimulation exerted a positive ino- 
tropic effect but tended to impair circulatory 
performance, lowering cardiac output and ar- 
terial pressure. On the other hand, when ap- 
plied during barbiturate-induced myocardial 
depression, paired electrical stimulation ele- 
vated cardiac output by an average of 129 per- 
cent, increased mean arterial pressure by 34 
mm. Hg, and lowered mean right atrial pres- 
sure by 2.4 mm. Hg. The effects of paired elec- 
trical stimulation were compared to those of 
acetylstrophanthidin and were found to be 
quite similar both in the failing and non-fail- 
ing heart. It was concluded that the effects of 
paired electrical stimulation are dependent on 
the circulatory state which exists at the time it 
is applied. 

Clinical Studies 

The maximum cardiac output response to in- 
tense exercise might be limited either by the 
pumping ability of the heart, or by extracar- 
diac factors limiting ventricular filling at a 
time when the ventricles are still capable of 
augmenting the cardiac output. Previous stud- 
ies have suggested the latter mechanism, since 
a relationship between total blood volume and 
maximum exercise capacity has been demon- 
strated; the increased exercise capacity pro- 
duced by training is associated with an aug- 
mented blood volume, and an impaired circula- 
tory response to maximal exercise has been pro- 



duced by decreasing blood volume. However, it 
is not known if cardiac output during maximal 
exercise can be elevated by acute expansion of 
blood volume. Accordingly, six normal men 
were studied at rest and during maximal tread- 
mill exercise before and after an acute infusion 
of 1000-1200 ml. of the subject's own blood. 
Transfusion increased resting central venous 
pressure from an average of 0.0 to 1.9 mm. Hg 
and cardiac output from 5.34 to 6.81 L/min. 
(p<0.01). During exercise the augmented blood 
volume increased central venous pressure from 
an average of 1.2 to 8.6 mm. Hg, but cardiac 
output, which averaged 21.60 L/min. in the 
control studies, was not significantly changed 
following transfusion (21.62 L/min.); maxi- 
mum 2 uptake and the postexercise 2 debt 
were also unaltered. These results indicate that 
since total blood volume and ventricular filling 
pressure normally do not limit maximum car- 
diac output and 2 uptake, cardiac perform- 
ance during maximum exercise must be limited 
by the pumping ability of the heart. 

Left ventricular function has been analyzed 
in a variety of patients with and without car- 
diac dysfunction by assessing the cardiovascu- 
lar responses to supine muscular exercise on a 
bicycle ergometer. Left ventricular end-dias- 
tolic pressure was measured continuously and 
cardiac output and oxygen consumption were 
determined before and during exercise. A nor- 
mal pattern was established in 7 patients with- 
out left ventricular dysfunction and consisted 
of an exercise factor equal to or greater than 
600 ml./ 100 ml. V0 2 , a left ventricular end- 
diastolic pressure (LVEDP) during exercise of 
less than 12 mm. Hg, and little change or a 
decrease in LVEDP, which was accompanied 
in most instances by an increase in the stroke 
volume. This response was then compared with 
that observed in 14 patients studied after ste- 
notic or regurgitant malformations of the 
aortic valve had been corrected by valve re- 
placement with a Starr-Edwards prosthesis, 
and with that seen in 31 patients with various 
other cardiac lesions. 

In the majority of patients with mitral 
stenosis the pattern of left ventricular func- 
tion during exercise was considered to be nor- 
mal, and in 5 patients with aortic valve pros- 



NATIONAL HEART INSTITUTE 



93 



theses, a normal pattern was also observed. 
Among the remaining patients, including those 
with prostheses, aortic stenosis, or left ventri- 
cular myocardial disease, two types of abnor- 
mal performance of the left ventricle were 
identified. In some patients, an increase in 
LVEDP was accompanied by an increase in 
the stroke volume, a response considered con- 
sistent with abnormal left ventricular dynam- 
ics in which the ventricle appeared to utilize 
primarily the Frank-Starling mechanism. In 
the remaining patients, an increase in LVEDP 
was accompanied by no change or a fall in the 
stroke volume, a response considered indicative 
of depressed left ventricular function. Thus, in 
the majority of these patients, determination 
of the LVEDP before and during exercise per- 
mitted definition of normal or abnormal left 
ventricular function. The method described 
thus appears to supply a practical and useful 
means of evaluating the functional status of 
the left ventricle in patients with and without 
myocardial dysfunction. 

Although a considerable body of information 
is available concerning the circulatory response 
of patients with heart disease to exercise in 
the supine position, little is known about the 
circulatory response of these patients to exer- 
cise in the upright posture. Since many of the 
symptoms of cardiac patients occur during 
exertion in the upright posture, it was consid- 
ered that a thorough understanding of the 
hemodynamic alterations occurring during 
mild to intense upright exercise would be of 
physiological and clinical importance. It was 
also hoped that a more reliable means of eval- 
uating the relative impairment of cardiac 
function could be obtained. Accordingly, pa- 
tients with various types of heart disease and 
normal subjects were studied during mild and 
intense levels of treadmill exercise. A Cournand 
catheter was introduced into the pulmonary 
artery in order to measure pulmonary arterial 
pressure and to obtain samples of mixed venous 
2 content. Cardiac output was measured by 
the Fick principle, and oxygen consumption 
(V0 2 ), arterial blood pressure, and the elec- 
trocardiogram were continuously recorded. 

When cardiac output was plotted against V0 2 
in the conventional manner, considerable over- 



lap was found between the two groups both in 
the absolute values of cardiac index and V0 2 , 
as well as in the slope of the line relating these 
two variables. Since the "exercise factor" de- 
scribes the slope of the line relating cardiac 
output to V0 2 , these results demonstrated that 
the "exercise factor" is an insensitive method 
for assessing the degree of cardiac impairment. 
On the other hand, if the cardiac index is 
plotted against the pulmonary artery (PA) 2 
saturation, a clear difference in the response 
to exercise between normal subjects and pa- 
tients was evident. At lower levels of exercise 
(PA saturation > 40%) the cardiac output re- 
sponse of the two groups did not differ greatly. 
However, at levels of exercise which produced 
a PA 2 saturation of 35% or less, the circu- 
latory response of the patients contrasted dra- 
matically with that of normal subjects. It was 
found that at a PA 2 saturation of 30%, all 
normal subjects achieved a cardiac index 
greater than 7 liters/min, while no patient ex- 
ceeded a value of 5 liters/min. Thus, the level 
of the cardiac index at a PA 2 saturation of 
30% provides a new and sensitive index of 
cardiac function which may prove to be of con- 
siderable clinical value. 

A comparison of the hemodynamic responses 
to exercise in the supine and upright positions 
was undertaken in patients with varying de- 
grees of impaired cardiac function. In patients 
with insignificant cardiac defects, cardiac out- 
put, mixed venous 2 saturation, and pulmo- 
nary arterial pressure were slightly but con- 
sistently higher at any given level of V0 2 in 
the supine position as compared to the upright. 
In contrast, in the patients with significant 
cardiac impairment, much higher pulmonary 
arterial pressures occurred in the supine posi- 
tion than during upright exercise. It also ap- 
peared that there was less of a difference be- 
tween the cardiac output during exercise in 
the supine and upright positions in the pa- 
tients as compared to the subjects with insig- 
nificant cardiac disease. 

Two of the more prominent manifestations of 
imparied cardiac function are an inability to 
augment the cardiac output appropriately in 
response to increased metabolic demands, and 
a diminished abiilty to excrete Na, with re- 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sultant fluid accumulation and edema. Since it 
has recently been observed that the cardiac 
response to maximal and submaximal levels of 
exercise in both normal subjects and patients 
with various forms of heart disease is reduced 
by blockade of the beta-adrenergic receptors, 
it became of interest to determine if this im- 
pairment of hemodynamic performance is as- 
sociated with changes in the patterns of Na 
excretion. 

Sixteen subjects, 13 men and 3 women, rang- 
ing in age from 21 to 57 years were studied. 
Six of the men were normal volunteers; the re- 
mainder of the subjects had heart disease of 
various types. Beta blockade altered the pat- 
tern of Na excretion in all subjects, and this 
impairment of Na excretion could be divided 
into three grades of severity. The mildest degree 
of impairment, observed in normal subjects 
and patients with reduced cardiovascular re- 
serve, consisted of an alteration in the diurnal 
pattern of Na excretion, the total 24-hour Na 
excretion remaining unchanged. An interme- 
diate degree of impairment, observed in some 
patients with heart disease, but not in normal 
subjects, was manifest by retardation of the 
rate of increase of Na excretion in response 
to progressive increases in Na intake, although 
Na balance was ultimately achieved. The most 
serious degree of impairment, observed in only 
one patient with heart failure, consisted of 
progressive Na retention, resulting in increas- 
ing fluid accumulation and edema. Although 
the results of this investigation suggest that 
beta-adrenergic blockade will not cause gross 
Na retention in most cardiac patients, it is 
stressed that inhibition of cardiac sympathetic 
nerve activity can occasionally precipitate dan- 
gerous cardiac decompensation unassociated 
with prior Na retention and weight gain. The 
results of this study further demonstrate the 
importance, particularly in patients with im- 
paired cardiac function, of the support which 
the sympathetic nervous system provides to 
myocardial performance. 

Although there has been extensive investi- 
gation of the central circulation in patients 
with heart failure, little information is avail- 
able concerning possible abnormalities of the 



peripheral vascular beds in these patients. 
Furthermore, only a few of the several varia- 
bles of the regional circulations have been 
correlated both among themselves and with the 
variables of the central circulation, even in 
normal subjects. At rest, forearm blood flow 
was found to vary directly with cardiac output, 
forearm resistance varied directly with total 
systemic resistance, venous tone varied direct- 
ly with forearm resistance and inversely with 
cardiac output. Patients with heart failure al- 
ways had lower values for forearm flow and 
higher values of forearm resistance and venous 
tone, providing complete separation of the two 
groups in the above comparisons. Cold stimula- 
tion and leg exercise resulted in an excessive 
elevation of forearm resistance and venous tone 
in patients with heart failure as opposed to 
normal subjects. 

It has been established that the elevation of 
the lower extremities in supine patients with 
congestive heart failure results in a decrease in 
forearm blood flow, while in normal subjects 
an increase in flow occurs. The mechanism of 
this paradoxical response in congestive heart 
failure has not been elucidated. In studies in 
which arteriolar and venous tone of the fore- 
arm were determined by plethysmography 
means, it was found that leg raising resulted 
in arteriolar and venous constriction in heart 
failure while the opposite effect was seen in 
normal subjects. In addition, it was observed 
that local nerve block in the forearm abolished 
the alteration of forearm blood flow in both 
groups of individuals, indicating that the vaso- 
constrictor response in heart failure and the 
vasodilator response in the normal subjects 
was mediated through a reflex. Since it is 
known that rapid changes in atrial pressure 
are important in initiating the abnormal vaso- 
constrictor response to leg raising in heart 
failure, it is postulated that a critical incre- 
ment in right atrial filling pressure reflexly 
initiates the response and these findings sug- 
gest that stretch receptors exist in these low 
pressure areas of the central circulation. 

The reactive hyperemia response, that is, the 
increase in blood flow immediately after resto- 
ration of the circulation after a period during 
which arterial inflow is occluded, was com- 



NATIONAL HEART INSTITUTE 



95 



pared in normal subjects and in a large group 
of patients with congestive heart failure, uti- 
lizing a plethysmographic technique. It was 
observed that the peak level of reactive hyper- 
emia as markedly reduced in the patients 
with heart failure compared to the normal sub- 
jects. The magnitude of the reduction of the 
hyperemic response was found to be a function 
of the duration of the period of circulatory ar- 
rest in normal subjects, progressive increases 
in postischemic blocd flow occurring as the du- 
ration of ischemia was prolonged, but this was 
not observed in the patients with heart failure. 
The mechanisms responsible for this phenom- 
enon have not yet been elucidated, but are cur- 
rently under investigation. It is unlikely that 
increased sympathetic vasoconstrictor activity 
can be held responsible, since local vasodilator 
metabolites are known to be capable of over- 
riding neurogenic effects. It would appear that 
the heart failure state in some manner in- 
creases the rigidity of the resistance vessels, 
and their altered mechanical properties do not 
allow them to respond in a normal fashion to 
the vasodilator metabolites that accumulate 
during circulatory arrest. 

Studies on the Mechanism of Action of Digitalis 

The digitalis glycosides are unquestionably 
the most important drugs used in the treat- 
ment of heart failure. Their mechanisms of ac- 
tion have been studied in this Branch since 
its establishment and these investigations were 
continued during the past year. 

Several investigators have proposed that the 
positive inotropic effect of digitalis on the 
heart is due, at least in part, to nore- 
pinephrine (NE) released from cardiac NE 
stores by the glycosides. Since this pro- 
posal is based on the finding of a 
decreased inotropic effect of digitalis after 
depletion of cardiac stores of NE by re- 
serpine or dichloroisoproterenol, the possibility 
was considered that this finding was due to a 
direct action of these antiadrenergic drugs 
rather than the depletion of NE. To examine 
this question of the responses of papillary mus- 
cles depleted of NE by chronic cardiac dener- 
vation and those in which the NE depletion 



was produced by reserpine pretreatment were 
studied. The muscles depleted of NE by chronic 
denervation respond normally to oaubain and 
strophanthidin while those equally depleted of 
NE by reserpine exhibited a reduced inotropic 
response to these glycosides. It is concluded that 
cardiac NE stores are not essential for the pos- 
itive inotropic effect of strophanthidin or oua- 
bain on the heart and that reserpine may in- 
terfere directly with the inotropic action of 
these cardiac glycosides. 

While digitalis glycosides improve the fail- 
ing myocardium, their effects on the normal 
heart remain controversial. Newer methods de- 
veloped in this laboratory permitted resolution 
of this controversey. The effects of ouabain 
(0.01 mg/kg) on ventricular force-velocity 
relations were studied in six patients following 
corrective cardiac operations. A beat-to-beat 
analysis of ventricular force-velocity relations 
was performed by relating the velocity of 
movement of roentgenopaque markers previ- 
ously sutured to external surfaces of the ven- 
tricles and intraventricular pressure at con- 
stant ventricular dimensions. It was observed 
that ouabain always augmented myocardial 
contractility as reflected in the force-velocity 
relation. Velocity of shortening increased an 
average of 77 ±5% above control while intra- 
ventricular pressure rose by an average of 
23 ±6%. Despite this improvement in contrac- 
tility, no consistent changes in cardiac output 
were observed. Analogous changes in force- 
velocity curves were obtained when a cardiac 
glycoside was added to isolated papillary mus- 
cles removed from normal cats. It is concluded 
that the fundamental action of digitalis gly- 
cosides is to augment the contractile state of 
the human heart, whether normal or failing, 
but that in the absence of heart failure this 
improvement is not translated into an increase 
in cardiac output. 

There has been considerable dispute concern- 
ing the effects of digitalis on myocardial 2 
consumption (MV0 2 ) and efficiency. Analyses 
of previous data suggested that the interpreta- 
tion of the results was complicated by the 
changes in circulatory dynamics induced by 
the drug. Accordingly, the effects of acetyl- 
strophanthidin (Avg. dose = 0.26 cat units/ 



96 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



kg) were studied in 6 non-failing, canine, right 
heart bypass preparations in which heart rate, 
stroke volume, and mean aortic pressure were 
held constant. MV0 2 increased in all experi- 
ments, by an average of 2.56 ml/min, while 
calculated cardiac efficiency declined by an av- 
erage of 24.4% of control. Even though mean 
arterial pressure was held constant, the glyco- 
side reduced the integrated systolic tension by 
an average of 40% and the peak systolic ten- 
sion by an average of 18%, chiefly as a conse- 
quence of a small decline in left ventricular 
end-diastolic volume. However, the velocity of 
myocardial fiber shortening increased consid- 
erably, the peak left ventricular ejection rate 
rising an average of 36% and the peak ven- 
tricular dp/dt increasing by an average of 
82%. Acetylstrophanthidin did not alter MV0 2 
in two hearts which were studied in an identi- 
cal manner, but in which left ventricular end- 
diastolic pressure was initially elevated. 
However in those experiments the large fall 
in end-diastolic volume resulted in a 
marked fall in systolic tension. In ad- 
dition, in one experiment, following acetyl- 
strophanthidin the left ventricular end- 
diastolic pressure and consequently myo- 
cardial wall tension were re-elevated to 
near control levels by increasing stroke 
volume. Under these circumstances, with 
no change in myocardial wall tension, oxygen 
consumption was increased with digitalis. It 
is concluded that digitalis tends to increase 
MV0 2 , but that its strongly positive inotropic 
effect frequently results in a reduction of ven- 
tricular wall tension which tends to oppose and 
to mask this effect. It is proposed that the in- 
crease in MV0 2 is related to the increased ve- 
locity of contraction induced by this agent. 

It is generally assumed that patients with 
mitral stenosis are, like patients with other 
cardiac disorders, benefited by digitalis. As a 
result, digitalis therapy was instituted in most 
patients with mitral stenosis, regardless of 
rhythm. However, the major impairment of 
cardiac performance in these patients results 
from mechanical obstruction to blood flow 
rather than from imparled myocardial func- 
tion. Based on this consideration, it was hy- 
pothesized that digitalis would not benefit pa- 



tients with mitral stenosis in normal sinus 
rhythm. On the other hand, relatively high 
mean left atrial pressures occur in patients 
with atrial fibrillation and rapid ventricular 
rates as a result of a greatly abbreviated dias- 
tolic filling time. In these subjects it would be 
expected that digitalis, by increasing the atrio- 
ventricular functional refractory period and 
thereby decreasing the ventricular response, 
would improve cardiac performance. 

Seven patients with pure mitral stenosis in 
normal sinus rhythm were studied at rest and 
at mild to intense levels of treadmill exercise. 
A Cournand catheter was placed in the pulmo- 
nary artery for measurement of pressure and 
withdrawal of mixed venous samples for car- 
diac output determination by the direct Fick 
method. Arterial pressure, oxygen consumption 
(V0 2 ), EKG, and heart rate were continu- 
ously recorded. After control values were ob- 
tained at rest and during exercise, ouabain, 
0.01 mg/kg was administered intravenously 
and the study was repeated. Several patients 
were given digoxin orally, and the studies re- 
peated 4 to 7 days later. In 3 patients both the 
acute and chronic studies were performed. The 
hemodynamic response to exercise was as- 
sessed by comparing cardiac output, V0 2 , pul- 
monary arterial pressure, and mixed venous 
oxygen saturation before and after the admin- 
istration of digitalis. In patients with normal 
sinus rhythm all of the measured parameters 
of cardiac performance were essentially un- 
changed by either acute or chronic adminis- 
tration of digitalis. Thus, these findings do not 
support the position that digitalis favorably 
influences the circulatory dynamics of pa- 
tients with mitral stenosis and sinus rhythm. 

Studies on Circulatory Control of the 
Autonomic Nervous System 

The spcific role played by the autonomic 
nervous system in circulatory control has been 
of interest to investigators in this Branch for 
a number of years now. This interest has con- 
tinued, and studies carried out on the auto- 
nomic nervous system are included among the 
other headings of this report. In addition to 
the aforementioned investigations, four others 
were carried out during the past year. 



NATIONAL HEART INSTITUTE 



97 



It is generally agreed that norepinephrine is 
the mediator released at the sympathetic neu- 
roeffector junction. In addition, it is believed 
that the norepinephrine released from nerve 
terminals as a result of sympathetic nervous 
stimulation acts at the same sites as humorally 
transported norepinephrine. Furthermore, it 
is felt that blockade of neurally evoked vaso- 
constriction is as easily accomplished as block- 
ade of humorally mediated vasoconstriction. 

In order to study these aspects of neurotrans- 
mitter activity as they pertain to vasomotor 
control of the circulation, a canine preparation 
in which the skeletal muscle vascular beds of 
the hindlimb were perfused at a constant flow 
was utilized. Changes in perfusion pressure 
therefore reflected changes in vascular resist- 
ance of the limb. Reflex vasoconstriction in 
the limb was produced by bilateral carotid ar- 
tery occlusion and by hemorrhagic hypoten- 
sion. The changes produced by these reflex 
mechanisms were then compared with the 
changes in resistance brought about by the 
intra-arterial injection of norepinephrine. 

In the dogs that served as controls, both 
carotid sinus hypotension and intra-arterial 
norepinephrine caused large increases in vas- 
cular resistance. The other animals were pre- 
treated with phenoxybenzamine 15 mg/kg, a 
powerful alpha adrenergic blocking agent. In 
this group of animals, carotid sinus hypoten- 
sion once again produced a reflex vasoconstric- 
tion in the perfused hindlimb, although not to 
as great an extent as in the normal limb. In- 
tra-arterially administered norepinephrine, 
however, now produced an opposite effect, 
causing vasodilation. Thus, the pretreatment 
with phenoxybenzamine blocked the vasocon- 
striction normally produced by norepinephrine 
as a result of its alpha receptor stimulating 
properties and unmasked its beta receptor stim- 
ulating effects which produce vasodilation. This 
vasodilation could then be blocked by treat- 
ment with propranolol, a powerful beta-adren- 
ergic blocking agent, thereby confirming that 
it was indeed a beta effect which had been un- 
masked. 

These results indicate that humorally trans- 
ported norepinephrine and neurally released 
norepinephrine act to a large extent at spatially 



different sites. In addition, contrary to state- 
ments in the literature, it seems clear that the 
alpha receptors at the neuroeffector junction 
are not reached by blocking agents as effec- 
tively as are alpha receptors at other sites. Fi- 
nally, since reversal of reflex vasoconstriction 
was never seen, it seems reasonable to assume 
that the population of beta-receptors in the re- 
gion of the neuroeffector junction is smaller 
in comparison to the population of alpha re- 
ceptors than this ratio elsewhere. 

Since the demonstration by Marey in 1859, 
it has been known that heart rate varies in- 
versely with the arterial blood pressure. It is 
also firmly established that these changes in 
heart rate are mediated reflexly, the afferent 
arm of the reflex originating in the barorecep- 
tors located in the carotid sinus and aortic arch 
areas. It was felt that it would be of interest to 
define the relative roles of the two main baro- 
receptor areas in the control of heart rate. 

In nine dogs the carotid arterial circulation 
was isolated from the rest of the vascular bed 
and was perfused by a donor dog. Changes in 
arterial pressure could then be produced inde- 
pendently in the carotid sinus and in the aortic 
arch baroreceptor areas. It was found that both 
baroreceptor regions are important in the con- 
trol of heart rate, although the aortic arch 
areas often predominate. It was also shown 
that a positive input, produced by raising the 
perfusing blood pressure is a more adequate 
stimulus than the negative input of lowering 
perfusion pressure. Finally, as shown by large 
changes in blood volume in the trunk which oc- 
cur when carotid sinus pressure is raised, re- 
flexes originating in this area exert a profound 
influence on the volume of the peripheral vas- 
cular bed. 

The central nervous system controls heart 
rate by varying the impulse traffic in sympa- 
thetic and parasympathetic nerve fibers termi- 
nating in the sino-atrial node. Although there 
has been considerable interest in the mechan- 
isms by which the heart rate is altered in re- 
sponse to exercise, postural changes, and stim- 
ulation of the baroreceptors, the relative role 
of the two divisions of the autonomic nervous 
system in mediating these changes in rate in 
conscious man have not been clarified. Further- 



98 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



more, litle information is available concern- 
ing the manner in which the heart rate response 
to baroreceptor stimulation is modified dur- 
ing exercise. 

The relative roles of the efferent pathways 
whch mediate the heart rate response to su- 
pine exercise and tilting were investigated by 
observing the separate and combined effects of 
beta-adrenergic blockade and parasympathetic 
blockade on cardiac acceleration in normal sub- 
jects. From these studies it appears that in the 
supine resting state, parasympathetic restraint 
is the dominant influence on heart rate, and 
the accelerating effects of sympathetic stimu- 
lation are minor. The speeding of the heart in 
response to mild exercise appears to result 
largely from withdrawal of parasympathetic 
inhibition, since cardiac acceleration was 
found to be essentially unimpaired by sympa- 
thetic blockade but to be inhibited by para- 
sympathetic blockade and double blockade. At 
higher levels of exercise, however, cardiac ac- 
celeration must result in part from sympa- 
thetic stimulation, since sympathetic blockade 
reduces the augmentation of heart rate in com- 
parison to the control study. The finding that 
the increments in rate during heavy exercise 
are smaller after double blockade than after 
parasympathetic blockade alone further identi- 
fies the contribution of the sympathetic sys- 
tem. In contrast to light supine exercise, 
substantial speeding could still occur during 
tilting when the parasympathetic system was 
blocked. Thus cardiac acceleration in response 
to mild supine exercise appeared to depend pre- 
dominantly on parasympathetic withdrawal, 
whereas that produced by tilting involved a 
relatively greater degree of sympathetic stim- 
ulation. No alteration in baroreceptor sensitiv- 
ity was found on transition from rest to 
exercise, and at rest, baroreceptor induced 
alterations in heart rate are moderated by 
stimulation or withdrawal of either efferent 
system. However, during exercise, heart rate 
changes are primarily dependent on changes 
in the activity of the sympathetic nervous 
system. 

The presence of baroreceptors in the walls 
of the two atria has been postulated by many 
investigators. However, the importance of 



these proposed receptors in the reflex control 
of vascular resistance and venous tone in intact 
man has not been elucidated. An investigation 
was carried out which was designed to deter- 
mine the effects of stimulation of the atria, by 
electrically pacing the right atrium with a 
catheter electrode, on the vascular dynamics 
of the forearm, and on the total peripheral 
vascular resistance. In 8 patients with normal 
or near normal cardiovascular dynamics at rest, 
stimulating the atria at a rate above the basal 
rate resulted in a striking augmentation of 
forearm blood flow and reduction of calculated 
forearm vascular resistance, indicating that 
arterial dilatation had taken place. In addition, 
a decrease of venous tone was produced, indi- 
cating venodilatation. Thus, it appears that as 
postulated, there are receptors in the walls of 
the atria, the stimulation of which are capable 
of producing vasodilatation of the normal hu- 
man forearm. 

Improvement of Cardiac Diagnostic Methods 

The direct measurement of oxygen satura- 
tion of blood in the central circulation was 
made possible by the in vivo oximeter system 
utlizing fiber optics, as described in the 1965 
annual report. Although that catheter system 
permitted the rapid continuous and accurate 
measurement of oxygen saturation, its clinical 
usefulness was markedly limited by the neces- 
sity of introducing a second catheter to meas- 
ure pressure, to inject indicator, or to 
withdraw blood. Accordingly, attention was di- 
rectly at encouraging and aiding in the devel- 
opment of a catheter which incorporates both 
fiber optics and a lumen. This catheter is now 
being utilized in the precise diagnosis of cer- 
tain forms of congenital heart disease and in 
studying the oxygen saturation and blood pres- 
sure in the pulmonary artery during and fol- 
lowing exercise in patients with varying de- 
grees of impairment of cardiac performance. 

In patients with congenital heart disease, the 
simultaneous measurement of oxygen satura- 
tion and pressure is making possible a more 
rapid exploration of these variables within the 
central circulation. It has been used to localize 
precisely and to quantify both left-to-right and 
right-to-left shunts by oxygen saturation 



NATIONAL HEART INSTITUTE 



99 



changes at these sites. The catheter lumen also 
permits the measurement of pressure, the cen- 
tral injection of indicator for peripheral arte- 
rial sampling and the withdrawal of blood for 
in vitro measurements. The execution of these 
maneuvers with a single catheter has proven 
useful and the simultaneous and direct meas- 
urement of oxygen saturation and pressure has 
been found particularly valuable in diagnostic 
procedures. 

In normal erect subjects pulmonary blood 
flow is greater to the dependent zones of the 
lung than to the apices, and this pattern may 
be reversed in mitral stenosis. External scin- 
tillation scanning of intravenously adminis- 
tered I 131 labeled macroaggregated of human 
serum albumin (MAA) was employed to eval- 
uate distribution of pulmonary blood flow in 
the erect posture in 13 normal subjects and 35 
patients with mitral valve disease. The upper/ 
lower third ratio of blood flow (U/L) to the 
right lung averaged 0.43 in normals and was 
significantly greater (1.01) in patients with 
mitral valve disease (p< 0.001). In the absence 
of heart failire U/L did not exceed 0.65 until 
MLAP exceeded 14 mm. Hg and was less than 
0.80 whenever MLAP was less than 15 mm. 
Hg. The correlation between U/L and MLAP 
was significant (r-Z 0.91, p< 0.001) and 
from the regression equation (MLAPZ = 14.0 
U/L5.32), MLAP could be predicted to 
within 5 mm. Hg at the 95% confidence level. 

It is concluded that the close correlation be- 
tween the U/L ratio and the mean left atrial 
pressure indicated that it is possible to pre- 
dict the latter within reasonable limits in pa- 
tients with mitral valve disease by means of 
simple lung scanning. The method has been 
found especially useful in the screening of 
asymptomatic patients with the clinical find- 
ings of mitral valve disease, and, in the preop- 
erative study of patients so ill that left heart 
catheterization was considered to be unusually 
hazardous. Scanning is also of value in deter- 
mining whether the pulmonary venous pres- 
sure is elevated in patients with known severe 
pulmonary arterial hypertension, so that the 
presence of potentially correctible lesions such 
as mitral stenosis or cor triatriatum may be 
detected. 



The technique is safer, produces less discom- 
fort, and provides more quantitative data con- 
cerning the distribution of pulmonary blood 
flow than either pulmonary arteriography or 
differential bronchospirometry. The necessary 
equipment for the application of the scanning 
method is far simpler than that required for 
the radioactive gas techniques and it is readily 
available. 

Scintillation scanning of the lungs after ad- 
ministration of I 131 labeled macroaggregates 
of human serum albumin has also been em- 
ployed to delineate the patterns of pulmonary 
blood flow in patients with congenital heart 
disease. It was found that in patients with tet- 
ralogy of Fallot the blood flow through a sub- 
clavian-pulmonary arterial anastomosis is di- 
rected principally to the lung on the same side 
as the anastomosis and that the relative dis- 
parity in flows to each lung provides an in- 
dex of the efficacy of the anastomosis and of 
the development of pulmonary atresia. Also, 
the presence of anomalies that create pulmo- 
nary venous hypertension may be detected by 
demonstrating a reversal of the normal increase 
in perfusion from lung apex to base when the 
patient is in the erect posture. Avascular areas 
of the lung secondary to atresia or marked nar- 
rowing of the pulmonary arteries may also be 
detected by lung scanning. Right-to-left shunt- 
ing through a patent ductus arteriosus may be 
diagnosed if the accumulated radioactivity in 
the capillary bed of the feet exceeds that in 
the hands after intravenous injection of MAA. 

Clinical Studies 

Idiopathic Hypertrophic Subaortic Stenosis 

A detailed anatomic, hemodynamic, angio- 
graphic and clinical review of idopathic hyper- 
trophic subaortic stenosis (IHSS) was 
undertaken in relation to possible mechanisms 
responsible for the intraventricular pressure 
gradient that occurs in this disease. The fea- 
tures were considered in particular relation to 
the recent proposal that cavity obliteration, 
rather than obstruction, is responsible for the 
pressure gradient in these patients. Anatomic 
observations at necropsy and at operation have 
revealed "bars" or "bands" of hypertrophic 



100 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



muscle which appear to obstruct the left ven- 
tricular outflow tract; incision of these hyper- 
trophied areas of the interventricular septum 
appears to abolish a sphincter-like action of 
the ventricular outflow tract. Review of hemo- 
dynamic observations, using a flowmeter at the 
time of cardiac surgery, has revealed that ap- 
proximately 70% of the stroke volume is 
ejected during the time that a pressure gradi- 
ent exists. Angiograms were reviewed in an 
effort to clarify the location of the site of ob- 
struction, and it was concluded that a linear 
radiolucent area can frequently be identified, 
presumably at the point where the hypertro- 
phied septum impinges upon the anterior leaf- 
let of the mitral valve in late systole. Combined 
hemodynamic and angiographic measurements 
were made in 14 patients. It was 
shown that an abnormally elevated left ventri- 
cular systeolic pressure could be recorded in 
the inflow tract of the ventricle, while the tip 
of the recording catheter clearly lay within the 
pool of contrast medium. In addition, in many 
patients it was shown that when a catheter 
inserted into the left ventricle by the trans- 
septal route was withdrawn from the area of 
the inflow tract into the left atrium, it never 
traversed a zone of low systolic pressure, i.e., 
one in which left ventricular systolic pressure 
equaled aortic pressure. Indeed, the tracing al- 
ways reverted directly from an elevated left 
ventricular systolic pressure to left atrial pres- 
sure. These findings, together with clinical 
evidence, such as the presence of a systolic 
murmur in patients without mitral regurgita- 
tion and the marked reduction or elimination 
of the pressure gradient by operations de- 
signed to relieve obstruction, support the con- 
cept that obstruction plays a significant role 
in IHSS. 

It is now well established that the discrete 
forms of obstruction to left ventricular out- 
flow remain constant during hemodynamic in- 
terventions, and that striking variations in the 
severity of obstruction may be induced in 
IHSS by a variety of physiologic and pharma- 
cologic stimuli. The effects of changes in heart 
rate on the severity of obstruction in IHSS 
have not been clarified. However, since ventri- 
cular volume is reduced and the contractile 



state of the myocardium is improved when 
cardiac rate increases, the possibility was con- 
sidered that this intervention might intensify 
the obstruction in IHSS. The effects of increas- 
ing heart rate with atropine were determined 
in patients with IHSS and compared to the 
responses in patients with discrete forms of ob- 
struction to left ventricular outflow. It was ob- 
served that an increase in heart rate, presum- 
ably through a decrease in left ventricular 
end-systolic volume, increased the trans-left 
ventricular pressure gradient and the severity 
of obstruction in IHSS. In patients with dis- 
crete obstruction the gradient was reduced and 
the orifice size was unaltered by increased 
heart rate. Therefore, the importance of heart 
rate in IHSS has been defined and, in addition, 
a useful diagnostic test for the differentiation 
of dynamic and fixed obstruction to left ven- 
tricular outflow has been provided. 

Changes in body position have been shown 
to influence the degree of obstruction in IHSS. 
Tilting to the head-up position augments the 
left ventricular outflow tract gradient and tilt- 
ing to the head-down position has been found 
to ameliorate the obstruction. Although the 
resting apex cardiogram of patients with IHSS 
is often quite characteristic of this condition, 
frequently physiological and pharmacological 
stimuli must be used in order for the charac- 
teristic second systolic wave to appear. A study 
was undertaken to determine if the apex car- 
diogram recorded during tilting could be used 
as a simple diagnostic procedure to establish 
the diagnosis of IHSS. Eight of 17 of the pa- 
tients studied had a supine apex cardiogram 
characteristic of IHSS. Characteristic changes 
of IHSS could be elicited in 6 of the remain- 
ing 9 patients by use of the tilting maneuver. 
It is concluded that the use of the apex cardio- 
gram during tilting is a simple and valuable 
adjunct in the diagnosis of IHSS. 

Exercise is a potent stimulus to angina pec- 
toris in certain patients with IHSS. Exercise 
has been shown to activate cardiac adrenergic 
receptors and to increase both the exercise and 
post-exercise outflow tract gradients. Beta- 
adrenergic blockade has previously been shown 
to cause a decrease in the left ventricular out- 



NATIONAL HEART INSTITUTE 



101 



flow tract gradient during exercise when cam- 
pared with the pre-blockade exercise state. An 
investigation was undertaken to determine if 
beta-adrenergic blockade induced by oral pro- 
pranolol would be of benefit in preventing ex- 
ercise induced angina pectoris in patients with 
IHSS. Five of six patients with exercise-in- 
duced angina pectoris were improved on the 
days when propranolol was administered. 
These 5 patients walked an average of 10.5 
minutes on the days when propranolol was ad- 
ministered in contrast to 6 minutes when 
placebo was administered. These differences 
are highly significant statistically, and pro- 
pranolol appears to be a valuable drug in the 
treatment of IHSS. 

Other Clinical Studies 

Only limited information is available con- 
cerning the symptomatic and hemodynamic 
results of operative correction of atrial septal 
defect (ASD) in older patients. Moreover, in 
patients with ASD beyond the fourth decade, 
in whom the lesion is commonly associated with 
significant symptomatic disability, the criteria 
for selection for operative treatment have not 
been well established. Accordingly, the clinical 
and hemodynamic findings in 48 patients with 
ASD over 40 years of age have been reviewed. 
The majority of patients (92%) reported sig- 
nificant symptomatic disability, and at cardiac 
catheterization they exhibited mild to moder- 
ate pulmonary arterial hypertension in the 
presence of a moderate to large left-to-right 
shunt. Thirty-four patients underwent opera- 
tive correction of the ASD, since 1960, how- 
ever, only one has died as the result of opera- 
tion. Following operation, all 12 Class III and 
IV patients, and 10 of 14 Class II patients were 
substantially improved. Postoperative cardiac 
catheterization revealed a substantial reduc- 
tion in pulmonary arterial pressures in 15 of 
the 16 patients in whom it was present pre- 
operatively. These results suggest that in the 
presence of a large left-to-right shunt, with or 
without moderate pulmonary arterial hyperten- 
sion, age should not contraindicate operative 
correction of an ASD when symptomatic dis- 
ability is present. 



Two studies have been carried out on the 
function of the left atrium in patients with mi- 
tral valve disease. In the first, the determi- 
nants of the height of the left atrial contrac- 
tion wave (a wave) were analyzed in 53 
patients with pure mitral stenosis and sinus 
rhythm. Ratios of the height of the a wave to 
the v wave, the height of the a wave to the 
mean left atrial pressure, and the a wave pulse 
pressure to the mean left atrial pressure were 
related to a number of variables which are re- 
lated to the severity of the mitral stenosis, i.e., 
to the mean mitral valve pressure gradient, 
the mean left atrial pressure, the mean pulmo- 
nary artery pressure, and the left atrial size, 
estimated roentgenographically. It was ob- 
served that the relative height of the a wave 
varies inversely with the hemodynamic sever- 
ity of the mitral obstruction, indicating that 
as the disease process progresses, left atrial 
contraction may become weaker and make a 
smaller contribution to left ventricular filling. 

The occurrence of an atrial gallop sound is 
an unusual finding in patients with mitral re- 
gurgitation. The development of atrial fibrilla- 
tion in many patients with severe mitral re- 
gurgitation precludes the occurrence of an 
atrial sound and standard tests and reviews 
have not called attention to this finding even 
in patients with mitral regurgitation and sinus 
rhythm. The frequency of atrial gallop sounds 
was determined in a group of 51 patients with 
pure mitral regurgitation in whom the diag- 
nosis was established at open operation. Nine 
patients demonstrated atrial gallop sounds and 
all nine had ruptured chordae tendineae. None 
of six patients with primary rheumatic mitral 
valvular regurgitation and sinus rhythm dem- 
onstrated this finding. A history of rheumatic 
fever was uncommon in patients with ruptured 
chordae tendineae but a history of subacute 
bacterial endocarditis was common in both 
groups of patients. The duration of a history 
of a heart murmur and of symptoms were 
shorter in patients with ruptured chordae ten- 
dineae. The left atrial chamber was generally 
smaller in patients with ruptured chordae ten- 
dineae, and was evidently capable of contract- 
ing forcefully and causing a ventricular filling 
sound. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



From the clinical point of view it appears 
that in patients with mitral regurgitation 
whose cardiac disability is sufficiently severe 
to require mitral valve replacement, the find- 
ing of normal sinus rhythm should raise the 
suspicion of ruptured chordae tendineae. If, 
in addition, an atrial gallop sound is present 
the diagnosis of ruptured chordae tendineae is 
even more likely. 

Detailed preoperative and postoperative 
catheterization studies were carried out in 12 
patients with advanced mitral and aortic valve 
disease in whom surgical replacement of both 
valves was performed utilizing ball-valve 
prostheses. Since the prostheses effectively re- 
lieve the mechanical burden on the left ven- 
tricle, any residual impairment of circulatory 
function observed postoperatively can be at- 
tributed to an impairment of myocardial func- 
tion. It was observed that replacement of both 
valves resulted in restoration of normal or 
nearly normal hemodynamics, recorded in the 
resting state. However, studies of left ventricu- 
lar function during exercise, as assessed by 
the relationship between cardiac output and 
oxygen consumption, between cardiac output 
and left ventricular end-diastolic pressure, and 
between stroke volume and left ventricular end- 
diastolic pressure, indicated that significant 
abnormalities of myocardial function and dy- 
namics exist that were not alleviated by com- 
bined valve replacement. Therefore, it appears 
likely that replacement fibrosis secondary to 
underlying myocardial disease and coronary 
artery disease, or myocardial dysfunction con- 
sequent to a chronic severe hemodynamic bur- 
den, contributes to the preoperative disability 
in these patients, and, in addition, may pre- 
vent complete symptomatic recovery following 
insertion of valvular prostheses. This postop- 
erative impairment of myocardial performance 
appears to be more pronounced than that ob- 
served in patients with isolated replacement 
of either the mitral or aortic valve. 

The study of phasic arterial blood flow pat- 
terns in intact, unanesthetized man has not 
previously been possible because of practical 
limitations in flow-measuring techniques. 
Recent improvements in the design of electro- 



magnetic flowmeter circuits and the miniatur- 
ization of flow transducers, suggested the pos- 
sibility that such instruments might prove 
suitable for these measurements. The charac- 
teristics of phasic and instantaneous mean 
brachial arterial (BA) blood flow, measured 
with the electromagnetic flowmeter, have been 
investigated in 35 patients undergoing cardiac 
catheterization. Phasic BA flow patterns were 
characteristically altered in the presence of 
valvular aortic stenosis, aortic regurgitation, 
and in hypertrophic subaortic stenosis, when 
compared with patterns recorded in patients 
without left heart disease. All patients exhib- 
ited spontaneous, often large variations in 
mean BA flow at rest. During forearm muscu- 
lar contraction, striking reductions in BA flow 
occurred, and, during maximal muscular con- 
traction, actual cessation of flow was observed. 
Reflex forearm vasoconstriction consistently 
occurred during the Valsalva maneuver, and 
with unilateral carotid occlusion, while fore- 
arm vasodilatation was demonstrated during 
carotid sinus stimulation. Thus this technique 
has permitted the first description of the nor- 
mal pattern of phasic BA flow in unanesthe- 
tized human subjects, as well as the altera- 
tions in phasic flow contour accompanying 
various cardiac lesions. In addition, it has al- 
lowed investigation of rapid, transient altera- 
tions in peripheral arterial blood flow not pre- 
viously accessible to measurement. 

Although the overproduction of serotonin 
remains the hallmark of the carcinoid syn- 
drome, recently attention has been focused on 
other biologically active substances elaborated 
by carcinoid tumors. The effects of brady- 
kinin, epinephrine, and reflex stimulation of 
the sympathetic nervous system, were com- 
pared on the arteries and veins of the forearm 
in carcinoid patients and normal subjects 
utilizing a plethysmographic technique. In con- 
trast to the normal subjects, in the carcinoid 
patients, epinephrine produced flushes, pro- 
found arteriolar dilatation, and resulted in a 
rise of brachial arterial bradykinin and the 
kinin-forming enzyme kallikrein; sympathetic 
stimulation produced flushes, attenuated vaso- 
constriction, and elevated plasma bradykinin 
and kallikrein. Therefore, this study provided 



NATIONAL HEART INSTITUTE 



103 



evidence to implicate the kallikrein-kinin sys- 
tem in the genesis of the carcinoid flush. 

Idiopathic infantile hypercalcemia, a disease 
probably related to deranged vitamin D me- 
tabolism, may be a feature of the syndrome 
consisting of supravalvar aortic stenosis, men- 
tal retardation, and a peculiar "elfin" facies. It 
is not known if the observed multiple-system 
involvement is genetically determined or if any 
or all of the features are related to deranged 
maternal vitamin D metabolism or fetal vita- 
min D metabolism, or to a combination of the 
two. Accordingly, a study was undertaken to 
determine if vitamin D crosses the placenta, 
and to explore the relationship between hyper- 
vitaminosis D in the mother and the develop- 
ment of supravalvular aortic stenosis in the 
offspring. 

Pregnant rabbits were divided into groups 
so that in addition to the stock diet some were 
given large amounts of vitamin D throughout 
gestation. Vitamin D bioassays revealed 7 and 
8V2 times higher values of vitamin D in the 
mothers and the newborns, respectively, than 
in the controls, providing direct evidence of 
transplacental passage. A total of 14 anatomic 
abnormalities of the aorta was noted in 34 
offspring whose mothers received 1.5 million 
units of vitamin D throughout pregnancy. 
Definite similarities were noted between the 
aortic lesions in 8 rabbits and supravalvular 
aortic stenosis in the human. Thirty-five con- 
trol offspring showed no abnormalities of the 
aorta. It is concluded that the vascular toxic 
effects of vitamin D cross the placenta. It 
seems reasonable to suggest that an in utero 
derangement in vitamin D metabolism on the 
part of mother and/or fetus may be responsi- 
ble for supravalvular aortic stenosis, especially 
when the latter is associated with infantile 
hypercalcemia. 

Section of Clinical Biophysics 

The major activities of this section are con- 
cerned with exploration of basic mechanisms in 
the physiology of the lung, heart and vascular 
system. These objectives require strong pro- 
grams in instrument development and develop- 
ment of computational procedures as well as a 
strong laboratory program. These activities 



will be described under three major headings: 
Vascular Mechanics, Myocardial Mechanics, 
and Pulmonary Mechanics. 

Vascula?- Mechanics 

Several moderately long term studies were 
concluded this year which gave us much bet- 
ter insight into the gross features of vascular 
dynamics. If we are to understand the mech- 
anisms determining circulatory behavior, it is 
necessary to gain a clear picture of the me- 
chanical behavior of both the vessel wall and 
the underlying hydrodynamics. A large num- 
ber of special devices and instruments have 
been devised which have permitted isolation 
of various vascular segments in the larger ar- 
teries, such that discrete measurements of 
pressure, forces, flows, displacements, and di- 
mensions could be recorded continuously on 
multi-channel F.M. tape. Rather formidable 
data processing programs have been developed 
which permit examination of these simultane- 
ous variables for unique relationships among 
them. The major findings to date can be sum- 
marized in the following listing: 

1. The distensibility of the aorta decreases 
progressively along the aorta from the root 
to the bifurcation. 

2. The "elastic" component of this visco- 
elastic system dominates the mechanical be- 
havior. 

3. The vessel wall is incompressible. 

4. The vessel wall demonstrates anisotropic 
properties, i.e. the elastic constants are differ- 
ent in the three different directions. 

5. The longitudinal vascular tethering (the 
mechanical properties of the constraining tis- 
sues surrounding the vessel) have been meas- 
ured and can be represented by a linear mathe- 
matical model consisting of 1 elastic element, 
1 visco-elastic element, 1 viscous element, and 
an inertance. 

6. The harmonic content of both the pres- 
sure and the flow curves in the major arteries 
seldom exceeds about 10 cycles per second 
whereas the harmonic content of the pressure 
gradient and the time derivative of pressure 
usually exceeds 25 cps. 

7. The hydraulic input impedance to most 
vascular beds, including the pulmonary artery 



104 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and ascending aorta, are similar. The imped- 
ance spectrum is dominated by the constant 
term in each case. The time dependent terms 
decay rapidly with frequency. 

8. The estimation of instantaneous blood 
flow, using the pressure gradient technique, has 
been validated in physical models as well as in 
the living pulsating vessel. The mean devia- 
tion of computed flow from monitored flow is 
of the order of 5% in the physical model, and 
of the order of 10% in the living pulsating 
vessel. 

9. It has been established that the simple 
single degree of freedom model of pulsatile 
flow in a vessel segment proposed originally 
by this laboratory fits the experimental data 
as well as the more complicated and elaborate 
multidegree of freedom systems such as that 
suggested by Womersley. 

10. The relationship between the time de- 
rivative of pressure and flow is a very indirect 
one which depends on the lumped properties 
of the entire vascular bed and, thus, contrary 
to the assertions appearing in the literature, 
it cannot be assumed to bear a unique relation- 
ship to the pulsatile flow at a point in the 
vascular bed. 

11. Flow in the major arteries frequently 
can be shown to exceed the apparent critical 
Reynold's number, suggesting that arterial 
blood flow may be unstable or at the border- 
line between stable and turbulent flow during 
a significant portion of the cardiac cycle. 

12. Pressure or flow disturbances are pro- 
pagated along the arterial tree at a velocity 
which is dependent on both frequency and on 
position along the tree. Pressure moduli appear 
to increase with distance along the aorta in a 
manner consistent with the observation noted 
above that the arterial tree becomes progres- 
sively stiffer peripherally. The lower harmonics 
of both the pressure and flow curve demon- 
strate strong reflections. 

The foregoing observations have clarified our 
picture of circulatory dynamics and have 
produced a number of useful mathematical 
models whose validity has been shown to hold 
at particular points along the vascular system. 
It remains to be seen whether an appropriate 
integration of these "segmented" models can 



predict over-all system behavior under widely 
varying conditions. This question can be ap- 
proached only by "driving" the entire vascular 
bed with known flow functions and observing 
the degree to which such an integrated model 
will permit us to predict the corresponding 
pressure functions, vessel displacements, etc. 
A special hydraulic flow generator is being 
designed for this purpose. 

With the foregoing major features of cir- 
culatory dynamics reasonably well defined, we 
are now in a position to turn to questions con- 
cerning the "fine-structure" of the pressure- 
flow relationships such as measurement of in- 
travascular velocity fields. These studies will 
allow us to probe more deeply into mechanisms 
associated with vessel wall "injury," i.e. the 
processes related to the "wear and tear" of 
aging and disease. 

To this end we began developing specialized 
measuring techniques about two years ago in 
conjunction with the Engineering and Mechan- 
ics Staff at Catholic University of America. If 
we are to gain insight into the fine structure of 
pulsatile flow, it is necessary to develop a ve- 
locity sensing probe which can be used to ex- 
plore the entire flow field in an artery. Dr. Ling 
of Catholic University is a specialist in the 
area of "hot-wire anemometry," and over the 
past year has developed a needle blood-flow 
probe which will permit us to map these flow 
fields. Studies validating the fidelity of this in- 
strument are currently being concluded. The 
device is linear and has a dynamic response 
extending beyond the range of our monitoring 
capabilities. Calculated extrapolations indicate 
that it is useful far into the kilocycle per sec- 
ond range. Therefore, it should be possible not 
only to map the "time smoothed" velocity pro- 
files in major arteries but also to estimate the 
frequency spectrum and energy content of the 
associated turbulence when present. Prelimi- 
nary studies in animals using this device indi- 
cates that the velocity profile is not symmetri- 
cal in the descending thoracic aorta. 

The rather consistent topography of athe- 
roma distribution in the human aorta sug- 
gests that hemodynamic events may be asso- 
ciated with the genesis of these lesions. The 
localization of atheroma does not appear to be 






NATIONAL HEART INSTITUTE 



105 



related to patterns of pressure distribution, but 
rather, to areas where one might expect high 
shearing stresses and/or turbulence. This ob- 
servation has suggested a "working hypothe- 
sis" which can be used as a scaffold upon 
which to direct future research in this pro- 
gram. 

It is hypothesized that a flux of lipoprotein 
molecules is driven toward the subendothelial 
tissue space from the blood phase by a con- 
centration gradient. This gradient may be in- 
creased by raising the blood concentration of 
fat. The "diffusivity" of the lipoproteins will 
depend primarily upon their molecular weight 
and their temperature in laminar flow and 
will depend in addition upon the "eddy" vis- 
cosity in turbulent flow. 

Factors assumed to act against this concen- 
tration gradient and diffusivity are the me- 
chanical resistance of the endothelial cell wall 
and perhaps also an electrical field of opposite 
polarity. This potential field is assumed to em- 
anate either from a fixed charge system in 
the endothelial wall or from selectively ab- 
sorbed materials at the blood-wall interface. 

To test this hypothesis two pilot studies 
have been completed. The first was designed 
to produce localized turbulence (to increase 
diffusivity) in dog aortas. The objective was 
to measure an associated increased deposition 
of fat and/or endothelial damage in the area 
of turbulence. Turbulence was produced by in- 
troducing a polished aluminum cylindrical plug 
into the descending thoracic aorta of dogs. The 
plug contained 2 patent longitudinal grooves 
placed axially along opposite sides of the outer 
surface. The plug size was chosen to occlude 
the unstretched aorta. Blood flow thus was di- 
verted into the two longitudinal channels 
formed on three sides by the walls of the 
groove and on the fourth side by that portion 
of the vessel wall covering the groove. Dimen- 
sions and pressure drops were such that the 
Reynold's number of the flow in the groove 
was about 1000 (critical). Intravenous infu- 
sions of emulsified fat were then given and 
the animal sacrificed after two hours. Serial 
sections were made of the longitudinal strips 
of vessel which had formed one wall of the 



high-velocity flow-channel. These were exam- 
ined for endothelial cell injury and fat deposi- 
tion. It was somewhat surprising to note that 
in nearly half of the dogs marked endothelial 
damage occurred in the area of high-shear 
flow. In all animals a very high correlation 
was demonstrated between the calculated hy- 
draulic shearing stress on the endothelial wall 
and the associated degree of endothelial dam- 
age. At times the shearing stress appeared to 
erode completely the endothelial cells down to 
the basement membrane. Evidence of fat in- 
filtration roughly parallels the degree of cel- 
lular damage. Clear cut evidence of fat infil- 
tration under the basement membrane was 
found in about V4 of the animals, strongly 
suggesting that fat was actually driven from 
the blood phase into the subendothelial region. 
These studies are being repeated employing a 
better controlled "shearing stress" so that both 
moderate degrees of exposure as well as heavy 
degrees of exposure may be compared. 

The second type of preliminary study was 
to explore the magnitude of any electrical bar- 
rier that might exist at the endothelial sur- 
face. A device was made which could be 
placed around the living pulsating blood ves- 
sel such that the position of electrodes intro- 
duced through the wall could be sensed 
electrically and recorded. Silver-silver chloride 
electrodes were used with high impedance am- 
plifiers to sense the voltages across the blood 
vessel lumen as well as along the lumen. 

The intravascular voltage distribution was 
found to be constant throughout the blood 
phase at about — 2 millivolts with respect to 
the uninjured adventitial tissue of the vessel. 
At the intimal surface the voltage suddenly 
drops about 10 millivolts. Histologic examina- 
tion of the electrode site in these instances 
showed no endothelial damage. The inference 
to be drawn is that this voltage represents 
some sort of "fixed charged" system on the 
endothelial surface and is not an "injury po- 
tential." As the electrode is advanced into the 
wall the voltage becomes initially somewhat 
more negative; however, then becomes pro- 
gressively more positive usually climbing to a 
value somewhat positive with respect to the 



106 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



voltage in the blood phase and occasionally 
positive even with respect to the adventitial 
surface of the blood vessel. The voltage on the 
ventral aspect of the aorta is about 4 mv more 
negative than that on the dorsal aspect of the 
aorta. Voltage differences also could be de- 
tected along the intimal surface of the aorta 
but no consistent trend could be established. 
Thus, it appears that there is, indeed, an elec- 
trical field probably associated with some 
"fixed charged" system at the endothelial sur- 
face. The significance of this as a barrier to 
passage of lipoproteins remains to be evalu- 
ated. Its significance in repelling the nega- 
tively charged blood cellular elements is ob- 
vious. 

Myocardial Mechanics 

The problem of myocardial function has 
been approached by three different avenues. 
The first has been study of cat papillary mus- 
cle preparation in an effort to devise empirical 
laws which will allow us to characterize the 
mechanical behavior of small units of muscle 
fibers. The second has been anatomical study 
of specially fixed total heart preparations from 
which dimensions, fiber distribution, and ori- 
entation have been determined microscopically. 
The third has been to study the sequence with 
which the various muscle masses are recruited 
into contraction under varying conditions of 
pressure and flow. 

The papillary muscle studies were greatly 
hindered by Dr. Feigl's move to the University 
of Pennsylvania where he has only recently 
been able to resume them. Moreover, efforts to 
derive empirical mathematical models for the 
data that we already have on hand have been 
hindered by the repeated delays in activating 
the hybrid computer unit. A three component 
model to describe myocardial fiber behavior 
has been devised and checked out on the large 
Honeywell 800 computer, however, use of this 
approach is extremely slow and expensive, 
therefore, we have deferred further analysis 
of the data until the high-speed hybrid facility 
is available. 

The anatomical studies are approaching com- 
pletion and have demonstrated that the canine 



heart may be considered to have a gross con- 
figuration somewhere between a cylinder and 
an ellipsoid revolution. Microscopically we 
have obtained clear cut evidence of consistent 
patterns of fiber orientation in the left ven- 
tricular wall. The inner 40% of the wall con- 
tains fibers running primarily in an axial di- 
rection. The middle 50% of the wall has fibers 
running in a circumferential direction and the 
outer 10% again in the axial direction at about 
60° from the axis symmetry of the heart. 

Study of the sequence of contraction of the 
various muscle masses has indicated that a 
spectrum of behavior can be expected depend- 
ing upon the particular animal and the pres- 
sure presented to the ventricle. During systolic 
ejection about one-third of the hearts appear 
to contract primarily in a circumferential di- 
rection whereas one-third demonstrates pri- 
marily an apex to base contraction. The remain- 
ing third demonstrated various combinations 
of circumferential and longitudinal contrac- 
tion depending upon the phase of contraction. 
Attempts to correlate these modes of contrac- 
tion with the distribution of fibers and their 
orientation have been inconclusive primarily 
because of the small samples of anatomical 
data available. These studies will be extended 
such that a better sampling will be available 
and hopefully a relationship can be established 
between the sequence of contraction and the 
distribution and orientation of fibers in the 
muscle mass. 

Lung Mechanics 

The major activity over the past year in the 
area of pulmonary mechanics has been directed 
toward solution of two difficult practical prob- 
lems: automatic data processing techniques to 
handle the tremendous volumes of multi-chan- 
nel analog information necessary in defining 
the mechanical behavior of the lung, and sec- 
ondly, development of new instrumentation for 
the study of pulmonary mechanics in small 
animals, such as the rabbit. Although studies 
have continued in the area of mechanical auto- 
regulation of flow and flow through collapsible 
conduits, the pressing nature of the foregoing 
problems plus the continued unavailability of 



NATIONAL HEART INSTITUTE 



107 



the hybrid facility have made significant ad- 
vances in this latter area impossible. 

Analysis of the results of a study in pul- 
monary mechanics can present an overwhelm- 
ing problem of data reduction. In any mean- 
ingful or purposeful approach to these kinds 
of experiments, it is necessary to have the 
results analyzed from a particular experiment 
before proceeding to the next stage. Therefore, 
it is essential to streamline our methods of 
data analysis so that we may accomplish this 
objective. In a typical experiment 3, and us- 
ually more, variables are measured continu- 
ously with time as various prescribed respira- 
tory maneuvers are carried out. Although it 
is possible to record these data on a strip 
chart and then do a laborious graphic analy- 
sis, this frequently requires one man-week per 
experiment. This is obviously impractical, and, 
therefore, we have spent considerable effort 
developing processing techniques which will do 
these sorts of analyses automatically. 

General programs have been written and are 
now operational which will perform both edit- 
ing functions as well as data sorting routines. 
The editing routines compare the channels of 
data against certain editing criteria which may 
be specified, such as the absence of un- 
wanted noise, the adequacy of the prescribed 
maneuver, and so on. Once the data are edited 
to delete unwanted and redundant material, 
the data are then "sorted." 

"Sorting" consists of exploring the relation- 
ship between two variables at a time for se- 
lected constant values for all remaining vari- 
ables. If one has, in fact, captured all of the 
primary variables describing the system, then a 
meaningful relationship between the two se- 
lected variables will emerge. For example, in 
the case of pulmonary mechanics if one 
measures instantaneous flow, volume and pres- 
sure, one can ask for the relationship between 
pressure and flow for selected values of in- 
stantaneous volume. In this situation unique 
isovolume pressure flow curves emerge. At the 
experimenter's option, certain statistical fitting 
procedures then can be performed on the re- 
sulting data to allow the general form of the 
functional relationships to emerge immediately 
as smooth curves. 



Using these techniques it is now possible to 
perform an experiment one day and have the 
essence of the results displayed graphically the 
following day. The scientific economy of this 
is obvious. 

Standard techniques for measuring the var- 
iables for pulmonary mechanics in laboratory 
animals have not existed. This, therefore, has 
been our major effort in the area of pulmonary 
mechanics over the past year. A small body 
plethysmograph has been designed and fabri- 
cated in which instruments have been de- 
veloped for the accurate measurement of in- 
stantaneous respiratory flow, volume and pres- 
sure. The plethysmograph itself consists of a 
rigid plastic chamber in which it is possible 
to ventilate artifically an anesthetized and/or 
curarized animal. A positive-displacement elec- 
trical recording-spirometer continuously senses 
the volume changes of the animal. Instantane- 
ous flows are measured by a specially designed 
linear flow resistance device. The recording 
fidelity of these systems has been established 
in excess of 20 cps. Thus, adequate techniques 
are now available for the measurement of in- 
stantaneous flow and volume. 

The measurement of intrathoracic pressure 
in small animals however is quite another 
story. Detailed studies of the relationship of 
intrathoraic and intraesophageal pressure 
have been carried out in an effort to establish 
the conditions under which the latter could be 
used as an estimate of intrathoracic pressure. 
The obvious physiologic merit as well as sim- 
plicity of the esophageal approach makes con- 
siderable effort in this direction worthwhile. 
It has been found that over the upper two- 
thirds of the vital capacity the esophageal 
route is adequate for most purposes. However, 
there are a number of notable exceptions to 
this which must be evaluated. 

With the development of these better meas- 
uring techniques for small animals it is hoped 
that we can proceed to more detailed and bet- 
ter controlled studies of pulmonary mechanics 
in small animals as well as the development of 
suitable experimental animals for the study of 
the pathogenesis of chronic obstructive pul- 
monary disease such as emphysema. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



SURGERY BRANCH 

Clinic of Surgery 

The investigative efforts of the Surgery 
Branch have, as in past years, principally re- 
lated to studies of normal and abnormal cir- 
culatory physiology, particularly in relation to 
the methods and results of operative treatment 
in patients with congenital or acquired heart 
disease. 

A major proportion of patients referred to 
the Surgery Branch now require operations 
involving the use of prosthetic cardiac valves, 
and a number of clinical and laboratory in- 
vestigations have centered about the general 
problems associated with intracardiac prosthe- 
ses. The principal late complication which ac- 
companies the use of all presently available 
prosthetic valves is the formation of thrombus 
on them, and subsequent embolization. The 
anatomy of the calf heart and coagulation 
mechanisms of the calf closely resemble those 
of man, and various means of inhibiting 
thrombus formation on valves have been 
studied in this species. Standard Starr- 
Edwards valves universally collected thrombus 
in both the early and late postoperative period, 
while similar valves coated with graphite- 
benzalkonium-heparin seemed thrombus-re- 
sistant for one month, but later also clotted. 
In other calves with standard valves main- 
tained on warfarin, thrombus was prevented 
throughout the observation period of one year. 
The temporary thrombus-inhibiting effect of 
GBH coating is explained by other studies in 
which the coating was prepared with radio- 
active (H 3 ) heparin. With in vivo implanta- 
tion, 71% of the heparin was eluted from the 
surface within four hours, and more than 
95% of it had disappeared within 36 days. The 
Melrose prosthesis in the tricuspid position has 
been used as a standard means of promoting 
thrombus. All such valves clotted within 48 
hours in calves not given intravenous fluids 
postoperatively; animals given dextrose and 
saline survived for this period, but their valves 
revealed thrombus. In others given infusions 
of low molecular weight dextran during the 
first two days, 6 of 7 valves were thrombus- 
free at sacrifice. Studies are continuing to de- 



termine whether the effect of dextran in pre- 
venting thrombus formation is a specific one, 
or whether the effect is simply hemodilution. 
Initial observations indicate that the latter 
mechanism may be of principal importance. A 
controlled clinical study of the effectiveness of 
dextran after mitral valve replacement is un- 
derway, and most patients are treated with the 
agent for the 48-72 hours postoperatively. 

Seven patients with prosthetic mitral valves 
have died early with massive thrombosis of the 
prosthesis and atrium, and two others have 
died in the late postoperative period as the 
result of thrombus formation. A clinicopatho- 
logic study of the patients who died early 
indicated that the thrombosis probably re- 
sulted from a discrepancy between the size of 
the prosthetic valve and the size of the left 
ventricular cavity. In each, the muscular ven- 
tricular septum protruded into the cage of the 
valve, prevented full descent of the ball, and 
obstruction to left atrial emptying resulted. 
This observation has indicated the necessity 
for an unusually small Starr-Edwards valve, 
or a valve of the discoid type, in patients with 
mitral stenosis and small or normal left ven- 
tricular cavities. 

A new method for detecting intracardiac 
thrombus either pre- or postoperatively, has 
been studied in both animals and man. A rab- 
bit antibody specific for fibrinogen is labelled 
with I 131 and administered intravenously. The 
compound is concentrated where thrombus is 
in contract with blood. In dogs in which left 
atrial thrombi were produced experimentally, 
the radio-activity of the thrombus was suffi- 
cient to permit its detection by precordial 
scanning. In preliminary clinical trials, spe- 
cific labelling of left atrial thrombi was 
achieved in two patients with mitral valve 
disease, and in another with a thrombus in 
the left ventricle. 

The design and durability of the Starr-Ed- 
wards prosthetic valve has been proved in more 
than five years of clinical application but, as 
noted, thrombus formation on it is frequent. 
Past experience has proved that thrombus and 
emboli were never associated with the use of 
prostretic valves constructed of Teflon fabric, 
apparently because the valves quickly became 






NATIONAL HEART INSTITUTE 



109 



covered with host tissue. This principle has 
been applied to rigid Starr-Edwards valves, 
all metal parts of which were covered with a 
loosely woven fabric. Within six weeks of im- 
plantation all were covered with glistening 
tissue and none revealed thrombus. Continu- 
ing observations must be made to determine 
whether excessive tissue growth may result in 
dysfunction of the valve. Initial assessments 
have also been made of a new type of pros- 
thetic valve which may have advantages over 
those currently available. The valve consists 
of a ring-like body within which a stream- 
lined disc is held by an eccentric hinge. The 
opening angle of the valve can be controlled 
by the aerodynamic profile of the disc, and 
flow through it is largely laminar. Initial an- 
imal implantation is encouraging, and the 
valve will be subjected to detailed in vivo test- 
ing within the coming year. 

A major problem which may complicate the 
course of any open operation on the mitral 
valve is associated aortic regurgitation, and 
methods of detecting the aortic regurgitation 
before operation were assessed retrospectively 
in 156 patients with mitral valve disease. No 
troublesome regurgitation occurred when an 
aortic diastolic murmur was absent, but when 
such a murmur was present neither its in- 
tensity, the pulse pressure, nor the diastolic 
arterial pressure indicated the severity of the 
leak. The magnitude of regurgitant flow could 
be determined accurately only by cineaortog- 
raphy and this study is, therefore, indicated in 
every patient with mitral valve disease in 
whom a blowing diastolic murmur is heard. 

Abnormal left ventricular pressure pulses 
have previously been observed in patients with 
severe mitral regurgitation, indicating an ab- 
normal pattern of ventricular ejection and 
aortic flow. Instantaneous aortic flow was 
measured at operation in patients with severe 
mitral regurgitation before and after valve 
replacement, and in dogs in which mitral re- 
gurgitation of controlled magnitude was pro- 
duced. The pattern of aortic flow during mitral 
regurgitation was abnormal, and was charac- 
terized by an abbreviated systolic ejection per- 
iod and time to peak flow, an increase in peak 
flow and mean ejection rate, and increases in 



peak acceleration of flow and the volume 
ejected during the first half of systole. Normal 
flow patterns were recorded in the patients 
after mitral valve replacement. 

Another clinical study was made of the ef- 
fectiveness of operative treatment of atrial 
septal defect. Data were available in 175 pa- 
tients who were catheterized, operated upon, 
and followed thereafter. Six patients, four of 
whom were older than 40 years and were in 
congestive heart failure, died. Of 154 pa- 
tients studied postoperatively, all but 11 were 
proved to have complete abolition of the left- 
to-right shunt. A number of patients had se- 
vere pulmonary hypertension postoperatively, 
and with few exceptions elevated pulmonary 
arterial pressure and resistance persisted, even 
though the defect was closed. Residual pul- 
monary hypertension rarely caused symptoms, 
however. 

Approximately one-third of patients in 
have both clinical and hemodynamic evidences 
of severe tricuspid regurgitation, and in 
some clinics tricuspid valve replacement is 
frequently performed at the time of mitral 
replacement. Twenty-nine patients who had se- 
vere tricuspid regurgitation at the time of mi- 
tral valve replacement were managed conserva- 
tively; 24 had no operation on the tricuspid 
valve, and in five a tricuspid annuloplasty was 
performed. The operative mortality in this 
group (17%) did not differ from that in pa- 
tients without tricuspid regurgitation, and 
late clinical and hemodynamic studies re- 
vealed spontaneous regression of the tricuspid 
lesion as heart size and right ventricular pres- 
sure decreased. The experience indicates that 
tricuspid regurgitation which occurs in asso- 
ciation with mitral valve disease is usually of 
functional nature; the valve will become com- 
petent as the size of the heart decreases and 
tricuspid replacement is seldom indicated. 

The effects of tachycardia, induced by atro- 
pine, were assessed in patients with discrete 
aortic stenosis and in others with IHSS. With 
rate increases, the systolic pressure gradient 
was unchanged or fell in patients with fixed 
obstruction, while in those with IHSS the 
gradient always increased, and the calculated 
orifice area fell. It appears that the dimensions 



110 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of the left ventricle decrease with tachycardia 
in patients with IHSS, and that the adminis- 
tration of atropine may prove a useful provoca- 
tive test in patients with this form of muscu- 
lar subaortic obstruction. 

The usefulness of the left atrial pressure 
pulse in assessing the severity of mitral ste- 
nosis was determined in 53 patients who were 
in regular sinus rhythm. It was observed that 
the height of the atrial a wave varied inversely 
with the hemodynamic severity of mitral ob- 
struction, suggesting that in advanced stages 
of mitral stenosis left atrial contraction is 
weaker and contributes relatively less to left 
ventricular filling. Certain patients, who are 
operated upon after long periods of congestive 
heart failure do poorly postoperatively, pos- 
sibly because myocardial norepinephrine stores 
are depleted. An attempt was made to identify 
such patients by preoperative studies of leg 
vascular resistance, determined after ipsilat- 
eral femoral arterial injections of tyramine 
or norepinephrine. Resistance was twice as 
high in patients who had been in failure as 
in asymptomatic ones, and the norepinephrine 
dose-response curve was distinctly steeper in 
the heart failure group. Each patient with 
heart failure was proved to have exceptionally 
low cardiac norepinephrine content. 

Several laboratory investigations have been 
related to cardiac arrhythmias and anti-ar- 
rhythmic drugs. It is recognized that patients 
in atrial fibrillation who are receiving digitalis 
may show evidence of digitalis toxicity follow- 
ing restoration of regular rhythm by counter- 
shock. This clinical observation suggested a 
study of the effects of atrial fibrillation on 
digitalis tolerance. In dogs, the toxic dose of 
acetylstrophanthidin was determined on suc- 
cessive occasions by titration. Later, and in 
the same animals, atrial fibrillation was pro- 
duced by atrial pacing and acetylstrophanthi- 
din titration repeated. The animals tolerated 
10-15 percent more of the drug before develop- 
ing toxic manifestations during atrial fibrilla- 
tion. In other dogs some hemodynamic effects 
of atrial fibrillation were assessed. When fibril- 
lation was induced and ventricular rate was 
not controlled, systemic arterial pressure and 
cardiac output fell, and atrial pressure in- 



creased. In other animals measurements were 
made during atrial fibrillation and compared to 
those obtained during paced regular sinus 
rhythm at the same ventricular rate. Arterial 
pressure remained unchanged, but systemic 
flow was significantly lower during atrial fi- 
brillation than during regular rhythm at the 
same rate, an effect which can be attributed to 
impairment of atrial transport function. 

When ventricular tachycardia was induced, 
either by digitalis intoxication or by an ex- 
ogenous electrical pacemaker, the rate could 
be slowed in every animal by paired or cou- 
pled pacing. In these situations, ventricular 
tachycardia originated from a single ectopic 
focus. In other animals ventricular arrhyth- 
mias were produced by ligation of coronary 
artery branches, and multifocal ventricular 
contractions resulted. With this arrhyth- 
mia neither paired nor coupled pacing was 
effective, and either usually caused ventricu- 
lar fibrillation. 

Both lidocaine (Xylocaine) and procaine 
amide (Pronestyl) are widely used to control 
ventricular arrhythmias, but their effects on 
the contractile function of the heart have not 
been documented. In dogs in which heart rate, 
aortic pressure, and stroke volume were con- 
trolled, contractile force and left ventricular 
dp/dt were measured after these agents were 
administered. With lidocaine, either 1.5 or 3 
mg./Kg., contractile force fell 18% after three 
minutes, but returned to control levels within 
10 minutes. Either 6 or 12 mg./Kg. of pro- 
caine amide caused a progressive decrease in 
contractile force (maximum 15%) for 12 min- 
utes, and force remained depressed after 30 
minutes. Diphenylhydantoin (Dilantin) was 
evaluated in a similar manner. Contractile 
force decreased 32%, and in other animals de- 
pression of ventricular function curves was 
always observed. The drug also decreased total 
peripheral vascular resistance in steady ar- 
terial bow preparations. 

A continuing laboratory and clinical inter- 
est of this unit has been the development, 
evaluation, and application of various biologic 
adhesives. On the basis of previous experi- 
mental studies, methyl-2-cyanoacrylate mono- 
mer has been found a satisfactory but not 



NATIONAL HEART INSTITUTE 



111 



ideal hemostatic agent. It is now undergoing 
systematic use in patients to reinforce closures 
of sutured incisions in the heart and great 
vessels, and to control bleeding from raw dis- 
sected areas. The courses of these patients, 
and the pathologic findings in those that die 
will be analyzed. A new adhesive system, con- 
sisting of gelatin, resorcinol and formaldehyde 
(GRF) has been developed. It permits a strong 
bond to be achieved in the presence of moisture, 
and has a high initial viscosity. Original prepa- 
rations were found to be toxic to tissue because 
of excess formaldehyde, a defect largely cor- 
rected in more recent formulations. In the dog, 
GRF was used to close incisions in the ileum, 
and all animals survived. In other dogs it ef- 
fectively controlled bleeding from the bisected 
spleen, the transsected kidney, and the incised 
bladder. It is contemplated that GRF will be 
found suitable for clinical application within 
the year. 

Previous reports have described studies of 
the effects of morphine on the heart and pe- 
ripheral circulation, and recent experiments 
evaluated the mechanism of action of this drug 
in the treatment of pulmonary edema. Pul- 
monary edema was produced in dogs by tech- 
niques which simulated the clinical disorders 
in which pulmonary edema commonly occurs. 
Morphine was administered and, as pulmonary 
edema subsided, strikingly and parallel de- 
creases in pulmonary arterial flow and pres- 
sure, and left atrial and left ventricular end- 
diastolic pressures were recorded. It appears 
that the principal beneficial effects of mor- 
phine in pulmonary edema result from an in- 
crease in vascular capacitance, and an asso- 
ciated decrease in systemic venous return. 

Mitral stenosis is one of the most frequent 
cardiovascular malformations encountered in 
patients, but no satisfactory experimental 
counterpart has been previously devised. In 
dogs, a suture was passed from the tip of the 
left atrial appendage, through the mitral valve, 
and out the posterolateral wall of the left ven- 
tricle. A cylinder of plastic sponge was at- 
tached to the atrial end of the suture, and the 
through the mitral valve ring. In dogs studied 
in both the early and late postoperative per- 
iods, left atrial and pulmonary arterial pres- 



sures were uniformly elevated, and diastolic 
gradients between the left atrium and left 
ventricle were present in all dogs. Long-term 
studies of the effects of mitral obstruction on 
the pulmonary vasculature can now be made. 

Other techniques for producing and studying 
the pulmonary vascular changes which result 
from various congenital cardiovascular malfor- 
mations have been devised. The end of a lobar 
pulmonary artery is anastromosed to the dis- 
tal end of an adjacent pulmonary vein; the 
lobe is then perfused with oxygenated blood 
at low pressure. In other similar preparations 
lobes are supplied with oxygenated blood under 
high pressure, or venous blood under high pres- 
sure. In this manner the relative effects of 
oxygen content and perfusion pressure on the 
pulmonary vascular bed can be assessed. 

The strain gauge arch is widely utilized to 
evaluate ventricular performance but the hem- 
odynamic variables which may influence the 
indicated contractile force have not been sys- 
tematically investigated. In dogs, recordings 
of the contractile forces of both ventricles were 
made as heart rate, stroke volume, and after- 
load were separately altered. Force increased 
with heart rate, but did not change over a 
wide range of stroke volumes. At constant rate 
and stroke volume, left ventricular force was a 
direct function of aortic pressure, whether the 
ventricle was empty or doing external work; 
this response was shown to result from changes 
in coronary flow. The experiment indicat that 
measurements of contractile force afford an 
accurate indication of ventricular performance 
only when rate and afterload are kept 
constant. 

Several clinical studies have originated from 
the pathology section of the Branch. The kid- 
neys of 132 patients who died of valvular 
heart disease were examined, and four were 
found to have extensive renal hemosiderosis. 
Each patient had a fixed calcified aortic valve 
which was both stenotic and regurgitant. Renal 
hemosiderosis was discovered in the kidneys 
of seven additional patients who had had 
prosthetic aortic valves inserted. These find- 
ings indicate that either a prosthetic valve or 
a diseased aortic valve can be responsible for 
intravascular hemolysis and, although renal 



112 



ANNUAL REVIEW OE INTRAMURAL RESEARCH 



hemosiderosis was often severe, there was no 
evidence that impairment of renal function 
resulted. The hearts of 24 patients who had the 
carcinoid syndrome were examined, and 16 
were found to have carcinoid heart disease. 
In six, typical lesions were present in the left 
3,ide of the heart as well as the right. The 
presence of cardinoid heart disease was evi- 
denced before death only by the presence of a 
murmur, unless detailed diagnostic studies 
were performed. Sections of the livers of 123 
patients who died of valvular heart disease 
were examined and each was shown to have 
a brownish-yellow pigment in the centrolobu- 
lar hepatic cells. In none, however, was the 
pigment found to contain iron. In 24 patients 
of the group, however, selective stains were 
positive for hemosiderin and this finding was 
attributable to massive blood transfusions in 
the period immediately preceeding death. 

LABORATORY OF CLINICAL BIOCHEMISTRY 

Amine Biogenesis and Metabolism 

It is now generally accepted, as we originally 
proposed, that hydroxylation of tyrosine rep- 
resents the rate-limiting step in the production 
of norepinephrine. For this reason, inhibitors 
of this enzyme are far more effective in de- 
pleting tissues or norepinephrine than are in- 
hibitors of dopa decarboxylase or dpamine- 
iS-hydroxylase. Several classes of inhibitors 
of tyrosine hydroxylase have been found among 
tyrosine analogues and catechol derivatives. 
The former compete with tryosine the latter 
with the tetrahydropteridine cofactor. «- 
Methyl-tyrosine has proved to be a most po- 
tent inhibitor in vivo as well as in vitro. Using 
amethyl-tyrosine, it has been possible to show 
that when animals are subjected to stressful 
conditions such as severe exercise or exposure 
to cold, tissue levels of norepinephrine, even in 
brain, are maintained by virtue of a rapid re- 
synthesis. In other words stimulation of sym- 
pathetic tissues causes them not only to release 
norepinephrine but also to increase the rate of 
synthesis. These findings were corroborated by 
experiments with labeled tyrosine. It was 
shown that exercise and exposure to cold re- 
sult in the incorporation of 2 to 4 fold larger 



amounts of administered tyrosine- 14 C into 
norepinephrine. The procedures have no effect 
on the amount or specific activity of the free 
tyrosine- 14 C in the tissues. We have obtained 
some evidence of increased synthesis in isolated 
stimulated tissues. Other laboratories have 
obtained even more evidence for such an effect 
in isolated organ preparations. The mechan- 
ism for this increased synthesis is of interest. 
It appears that the increased synthesis is due 
to increased tyrosine hydroxylase activity. 
However, we have some evidence that neither 
exercise nor cold increase the absolute amount 
of the enzyme in tissues. It would appear, 
therefore, that the enzyme in vivo under nor- 
mal conditions is not operating at maximal 
capacity and that some factor or factors in- 
crease its activity. One possibility is that nor- 
epinephrine itself, which we have shown to be 
an inhibitor of tyrosine hydroxylase, controls 
the initial reaction by end-product inhibition. 
Several other laboratories have reported find- 
ings consistent with such a mechanism. 

We have obtained definite evidence that all 
three enzymes involved in norepinephrine syn- 
thesis are associated with a similar subcellu- 
lar fraction within tissues. We feel, however, 
that the homogenization and sedimentation 
techniques which were used are not adequate 
for unequivocal determination of intracellular 
localization. Because of this limitation, we 
have turned to fluorescent antibody methods. 
We have purified beef dopamine-/3-hydroxylase 
and prepared potent antibody to it in the rab- 
bit. A contract was awarded to Microbiological 
Associates to produce enzyme antibodies, pur- 
ify them and assist us in labeling them with 
fluorescent dye. 

Inhibition of tyrosine hydroxylase has been 
carried to the clinical level in joint studies with 
the Experimental Therapeutics Branch, a- 
Methyl-tyrosine was administered to patients 
and was shown to decrease the formation of 
norepinephrine. At doses of 1.5 to 3 gram per 
day about 70% inhibition was attained in a 
large number of patients with essential hyper- 
tension and pheochromocytoma. In the latter 
group, blood pressure was lowered to the nor- 
mal range as the excretion of norepinephrine 
and its metabolites fell to normal values. It 



NATIONAL HEART INSTITUTE 



113 



appears that a-methyl-tyrosine may become 
the treatment of choice for patients with ma- 
lignant pheochromocytoma. 

Another collaborative effort with the Exper- 
imental Therapeutics Branch concerns patients 
with phenylketonuria (PKU). Attempts will be 
made to answer two questions: (1) Is the 
small amount of conversion of phenylalanine 
to tyrosine which occurs in patients with PKU 
due to residual liver phenylalanine hydroxy- 
lase or is the latter enzyme completely lacking 
and the conversion carried out by the action 
of tyrosine hydroxylase in sympathetic nerves ? 
If the latter is the case, then a-methyl-tyro- 
sine, which specifically inhibits tyrosine hy- 
droxylase, should abolish conversion of phenyl- 
alanine to tyrosine in patients with PKU but 
have no effect on the conversion in normals. 
(2) Do the large amounts of phenylalanine in 
tissues of patients with PKU inhibit tyrosine 
hydroxylase sufficiently to disturb peripheral 
and central sympathetic activity? If so, then 
we may expect to find similarities between phe- 
nylalanine and a-methyl-tyrosine in their phar- 
macologic effects in patients without liver phe- 
nylalanine hydroxylase. Attempts are also be- 
ing made to produce animals deficient in liver 
phenylalanine hydroxylase activity by admin- 
istering p-chlorophenylalanine. 

Collagen and Hydroxypyroline 

Out laboratory was the first to demonstrate 
formation of collagen hydroxyproline in cell- 
free systems and to present evidence for hy- 
droxylation of peptidyl proline. Until last year 
several laboratories claimed that hydroxyla- 
tion occurred at the level of t-RNA-proline. In 
the last year these contrary claims have been 
answered and withdrawn and it is now clear 
from work in three other laboratories (Proc- 
kop, Lukens, Meister) that we were correct 
in the mechanism we proposed. Continuing 
these studies, we were able to show that all 
those tested of a number of tissues which form 
collagen are capable of accumulating a protein 
deficient in hypro- 14 C when incubated with 
proline- 14 C. This protein is formed when hy- 
droxylation is inhibited for any reason, e.g. 
lack of oxygen, chelation of Fe ++ , lack of as- 
corbic acid, destruction of proline hydroxylase. 



The protein can be extracted along with col- 
lagen by the use of neutral salt solutions or 
acetic acid and can be degraded to peptides by 
specific bacterial collagenase. More recently 
we showed that the solubilized newly formed 
hypro deficient "collagen" obtained from guin- 
ea pig granuloma, fetal rat skin and chick em- 
bryo can all be hydroxylated by chick embryo 
proline hydroxylase. The latter enzyme has now 
been demonstrated in soluble preparations ob- 
tained from rat liver, fetal rat skin, and guinea 
pig granuloma. Previously the chick embryo 
system was the only cell-free system in which 
proline hydroxylation could be demonstrated. 

Soluble proline hydroxylase, purified several 
fold, is stimulated by Fe ++ , ascorbic acid and 
an unknown dialyzable substance found in 
boiled tissue extracts. It dees not hydroxylate 
free proline or tripeptides containing proline. 
Proline, hydroxyproline, gly-pro-pro and gly- 
pro-hypro do not inhibit the hydroxylation of 
peptidyl-proline. This represents further evi- 
dence that smaller residues are not intermedi- 
ates in the hydroxylation. Two potent inhib- 
itors of peptidyl-proline hydroxylase are poly- 
proline-MW 10,000 and ( gly-pro-pro) n -MW 
5,000. The former has been shown to inhibit 
competitively with solubilized protein sub- 
strate. Attempts are being made to produce in- 
hibitors which are not only specific, but small 
enough to penetrate into cells and inhibit the 
enzyme in vivo. 

Studies on peptidyl proline hydroxylase have 
been time consuming because the method of 
assay took about two days. Several years ago, 
we had considered using tritium labeled pro- 
line to follow proline hydroxylation by oxida- 
tive displacement of a tritium atom and we 
persuaded Dr. Bernhard Witkop to prepare 3,4- 
tritio-proline from 3,4-dehydroproline. 



V 

H 



COOH 



v 

H 



COOH 



T+ + H 2 0?±THO + H+ 



In the crude cell-free chick embryo systems, 
we could not use this procedure because of 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



many interfering side reactions. However, 
Meister succeeded in using it in following hy- 
droxyproline formation in whole cell prepara- 
tions from guinea pig granuloma. Recently, 
we have been able to prepare soluble hydroxy- 
proline deficient substate, labeled with proline- 
3 H, for studies of proline hydroxylase. This 
material was purified and shown to contain 
traces of hydroxyproline- 3 H (less than 1% of 
the proline). On incubation with proline hy- 
droxylase we have demonstrated a stoichio- 
metric displacement of tritium from substrate 
proline into water which is collected by a rapid 
distillation procedure. The method is extreme- 
ly rapid, the blanks are essentially zero and 
the labeled substrate is stable indefinitely. 
With this procedure, we hope to purify the en- 
zyme, characterize it and develop useful in- 
hibitors. In collaboration with Dr. Arieh Ber- 
ger, we are looking into syntehtic polypeptide 
substrates and inhibitors. 

Actinomycin Biosynthesis 

Although attempts to obtain synthesis of this 
peptide antibiotic in cell-free systems have 
failed, it has been possible to obtain more 
information on the nature of the synthetic 
process. 4-Methyl-3-hydroxyanthranilic acid 
(MHA) has been identified as a normal inter- 
mediate in the formation of actinomycin. Pre- 
sumably this compound adds on the amino 
acid present in the pentapeptide chain. It has 
also been shown that the addition of D-valine 
to the medium results in the accumulation of 
MHA. This D-amino acid known to be an in- 
hibitor of antibiotic production, inhibited 
either the formation or the attachment of the 
pentapeptide chain. 

Other studies have shown that actinomycin 
inhibits the organism that produces it, sug- 
gesting that the production of the antibiotic 
may be a way by which the organism regu- 
lates its metabolism. Highly- labeled actinomy- 
cin has been produced and its distribution in 
tumor-bearing mice was investigated. 

Cobamide Dependent Methionine Synthesis 

The cobamide-dependent enzyme catalyzing 
methyl transfer from N 5 -methyl-folate to ho- 



mocysteine has been purified 200-fold. It is 
salmon-red in color and studies are being initi- 
ated on the binding of radioactive substrates 
to the enzyme. The unique function of S-adeno- 
sylmethionine (AMe) in this reaction has been 
investigated. It appears that AMe methylates 
the enzyme since other methyl donors (methyl- 
iodide, etc.) can replace Ame in the catalytic 
reaction. Studies are continuing in the forma- 
tion of boloenzyme. 

apoenzyme + cobamide »holoenzyme 

A specific enzyme is necessary for this reac- 
tion if the cobamide employed is either hy- 
droxy or deoxyadenosyl-B 12 . Attempts to pu- 
rify this enzyme as well as apoenzyme are in 
progress. 

Formation of Formyl-methionine sNRA 

The synthesis of N-formyl-methionine sRNA 
may be an important reaction in the initiation 
of protein synthesis. The enzyme that cata- 
lyzes the reaction 

N 10 formyl — THF + methionine sRNA — >N — formyl 
methionine sRNA + THF 

has been purified 2000-fold. The characteris- 
tics of the reaction are under study. The spe- 
cificity for the substrate resides in the sRNA 
molecule. Ethionine and norleucine, when at- 
tached to the methionine sRNA, are also for- 
mylated. 

Aromatic Hydroxylation 

5-Tritio-tryptophan has been synthesized 
and used to develop an assay for tryptophan 
hydroxylase. There is a marked isotope effect 
in a hydroxylase system from mast cells al- 
though none was observed in whole cells of 
Chromobacterium violaceum. 

Studies on the mechanism of ether cleavage 
by a microsomal system have shown that O 18 
is not incorporated into the phenolic product. 
This type of reaction does not involve a single 
displacement of the methoxy group by molecu- 
lar oxygen. 

Serine Metabolism 

A simple and sensitive method for the assay 
of serine hydroxymethylase has been developed. 



NATIONAL HEART INSTITUTE 



115 



It is based on the isolation of formaldehyde 
released as the dimedon derivative. 

2,3-Dihydroxybenzoylserine has been iso- 
lated as a metabolite of stationary cells of 
E. coli. It is synthesized by the following re- 
action: 

2,3 — dihydroxybenzoic acid + serine + 
ATP — >2,3 — dihydroxybenzoylserine. 

The enzymatic reaction and the role of this 
compound in the metabolism of E. coli are un- 
der investigation. 

Studies on Amino Acid Transport 

Three aminonaphthylalanines were prepared 
and shown to have fluorescence characteristics 
which were discernible in the presence of ani- 
mal and bacterial cells. Two of these amino 
acids were found to be concentrated by Sar- 
coma-37 ascites cells in a manner comparable 
to that of naturally occurring aromatic amino 
acids. The fluorescence of the amino acid after 
its active transport into cells did not differ in 
excitation spectrum, emission spectrum, quan- 
tum yield and temperature coefficient. Exami- 
nation with the fluorescence microscope indi- 
cated uniform distribution through the cyto- 
plasm. Furthermore, rupture of the cells re- 
leased the amino acids completely. By contrast 
atabrine, eosin and anilinonaphthalenesulfonic 
acid, which are known to bind to macromole- 
cules, exhibited changes in many parameters 
and were not released from the cell on rup- 
ture. Since intracellular concentration of the 
aminonaphthylalanines does not alter fluores- 
cence characteristics which are normally 
changed when a small molecule binds to a large 
one it is reasonable to assume that the amino 
acid exists in a free form. These findings rep- 
resent direct evidence that actively trans- 
ported amino acids exist free in the cell. 

Peptides and Peptide Linkages 

The major kinin in wasp venom has been 
purified and subjected to sequence analysis. 
Preliminary data indicate that glutamic acid 
is at the N-terminal end. The sequence of two 
amino acids remain in doubt (in parenthesis) 
but the following structure is fairly certain: 



glu (asp, thr) lys lys leu arg lys gly 

arg pro pro gly phe ser pro phe arg 



bradykinin 



sequence 



The demonstration of /?-aspartyl linkages in 
fibrin has been established. 

Brain Nucleic Acid 

Studies with rats in which the liver is ligated 
have shown that simple precursors such as as- 
partic acid, orotic acid and glucose do not 
yield appreciable labeling in brain RNA. These 
results agree with studies on brain slices which 
indicate that brain requires preformed nucleo- 
sides for its synthesis of RNA and presumably 
obtains them via the blocd from the liver. 

Bacterial Phenylalanine Hydroxylase and Its 
Cofactor 

Induced phenylalanine hydroxylase of Pseu- 
domonas sp. has been purified to a high de- 
gree. It has been shown to have an absolute re- 
quirement for Fe ++ but can be activated by 
many other metals. The pteridine cofactor re- 
quired for activity is also synthesized by the 
organism and it has been shown that it is de- 
rived from guanosine phosphate. Some of the 
intermediate enzymes required for synthesis of 
the pteridine have been isolated and studied. 

Other aspects of control of aromatic amino 
acid biosynthesis in Pseudomonas have also 
been investigated. 

Nitrogen Containing Lipids 

The significance of palmitylethanolamide is 
still not clear. On the one hand it is normally 
made and found in tissues. On the other hand 
its synthesis in in vitro preparations seems to 
represent an artifact formed by the reaction of 
ethanolamine with some form of "activated" 
fatty acid. The nature of this activated fatty 
acid in tissues remains to be determined. 

Enzyme Mechanisms 

Glutamic dehydrogenase, glyceraldehyde-3- 
phosphate dehydrogenase, tyrosine hydroxyl- 
ase and dopamine-/?-hydroxylase have been sub- 
jected to careful kinetic studies. Antibodies 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



have been prepared to some of these and the 
mechanism of interaction studied. 

EXPERIMENTAL THERAPEUTICS BRANCH 

A broad spectrum of research has been con- 
tinued over the past year with a major orienta- 
tion toward findings which may be of imme- 
diate or at least potential clinical significance. 
Data obtained will be considered under three 
headings: (1) Biochemistry and Pharmacology 
of Aromatic Amines, (2) Studies of Selected 
Proteins, and (3) Miscellaneous. 

Biochemistry and Pharmacology of Aromatic 
Amines 

It has been our impression that, with the 
exception of amines in urine collected follow- 
ing the ingestion of amine-containing foods, 
the urinary aromatic amines are of endogen- 
ous origin. Evidence has been presented in the 
literature, however, that the action of intesti- 
nal bacteria may contribute to urinary tyra- 
mine and tryptamine. Our studies on the effects 
of gluccse diets and intestinal sterilization 
have reconfirmed the conclusion that tyramine, 
tryptamine and metanephrine are of tissue ori- 
gin and reflect endogenous metabolism. Rather 
than a decrease in the excretion of tyramine 
and tryptamine during gut sterilization in pa- 
tients receiving a constant diet, a significant 
and consistent increase in excretion of these 
amines was observed. This latter finding has 
not been explained as yet. 

Previous work showed that a-hydrazino-his- 
tidine is a potent and selective inhibitor of his- 
tidine decarboxylase in animal systems both 
in vitro and in vivo. Clinical trials of the com- 
pound were initiated in three patients. It was 
quickly established that the drug is poorly tol- 
erated because of the development of emesis 
and further clinical use is not anticipated. Con- 
comitantly, a former member of the depart- 
ment has administered another histidine de- 
carboxylase inhibitor, 4-bromo-3-hydroxy-ben- 
zyloxyamine (NSD-1055), to patients with 
urticaria pigmentosa and has observed rapid 
and striking clinical improvement. Similar 
studies are planned in this department with 
NSD-1055 which is also a potent inhibitor of 



dopa decarboxylase and dopamine /3-oxidase. It 
was shown previously that up to 1.0 gm per 
day of this compound is well tolerated by hu- 
man subjects. 

A sensitive radioassay has been developed 
for measuring the activity of tryptophan hy- 
droxylase; the reaction catalyzed by this en- 
zyme appears to be the rate limiting step in 
the biosynthesis of serotonin. The assay was 
perfected using as a prototype the tryptophan 
hydroxylase from neoplastic murine mast cells. 
Preliminary attempts to study tryptophan hy- 
droxylase in mammalian brain tissue have in- 
dicated low levels of this enzyme in rabbit 
brain stem in some experiments. Unlike mast 
cell tryptophan hydroxylase, active prepara- 
tions of the brain stem were not stimulated by 
tetrahydropteridine or ferrous iron. The mast 
cell preparation is in use both here and else- 
where in screening programs for the discovery 
of the inhibitors of tryptophan hydroxylase. 
Such compounds should be useful in the treat- 
ment of patients with the carcinoid syndrome. 
First stage clinical testing is planned with one 
such agent, 3,4-dihydroxyphenyl-/3-propylacet- 
amide (dopacetamide), a potent inhibitor of 
tryptophan hydroxylase both in vitro and in 
vivo. 

Tryptophan deficiency has been suspected of 
causing African cardiomyopathy and serotonin 
of causing endomyocardial fibrosis. A number 
of studies of serotonin metabolism in trypto- 
phan deficient rats have been done with incon- 
clusive results. Peculiarly, such animals show 
a greater uptake of injected serotonin by the 
heart than do controls. Further studies are 
planned relative to reports in the literature 
that patients with African cardiomyopathy 
have elevated plasma serotonin concentrations 
and that tryptophan deficient rats have in- 
creased amounts of 5-hydroxyindole-acetic 
acid in the urine. 

Hydroxylation of tyrosine to dopa has been 
found to be the rate limiting step in the bio- 
synthesis of the catecholamines. In collabora- 
tion with members of LCB a number of stud- 
ies have been performed in laboratory animals. 
Rats, pretreated with the tyrosine hydroxylase 
inhibitor a-methyl-tyrosine («MPT) and then 
stressed by exercise on a treadmill or exposure 



NATIONAL HEART INSTITUTE 



117 



to cold (3° C), show a great reduction in brain, 
heart, spleen and adrenal catecholamines. 
There is little change with stress or aMPT 
alone. The findings are interpreted as indicat- 
ing increased rates of synthesis of norepine- 
phrine and epinephrine due to increased 
sympathetic nerve activity. The effect of sym- 
pathetic nerve activity on norepinephrine 
synthesis was evaluated more directly in a 
newly-designed, isolated nerve, perfused-heart 
preparation of the guinea pig. Nerve stimula- 
tion increased norepinephrine synthesis as 
judged by the difference in concentrations of 
the amine in the heart in the presence and ab- 
sence of «MPT. Thus far, however, it has not 
been possible to demonstrate an increased in- 
corporation of radioactivity into norepineph- 
rine from C 14 -tyrosine in the perfusing fluid. 
It may be necessary to add a monoamine oxi- 
dase inhibitor to prevent metabolism of the 
amine and to study the effect of varying con- 
centrations of tyrosine in the perfusing fluid. 
A number of tyrosine hydroxylase inhibitors 
have been compared for their catecholamine 
depleting potency in vivo in the hope that at 
least on compound in addition to aMPT might 
be available for clinical studies. One possibil- 
ity is L-3-iodo-a-methyl-tyrosine which is a 
more effective inhibitor than «MPT. Another 
is a-methyl-phenylalanine which is about as po- 
tent as aMPT and has the advantage of a much 
greater solubility in water. Clinical studies 
with aMPT have been concerned with its ab- 
sorption, metabolism, chemical effects and 
pharmacologic effects in patients with pheo- 
chromocytoma, and essential hypertension. 
About 30 patients have been studied, 17 of 
whom had pheochrcmocytoma. In oral doses of 
400-400 mg/day of aMPT, inhibition of cate- 
cholamine synthesis was shown as indicated by 
the urinary excretion of catecholamines and 
their metabolites. Reductions of synthesis up 
to 60-90% were achieved by the larger doses 
in both types of patients. In cases of pheochro- 
mccytoma reductions of catecholamine synthe- 
sis were accompanied by marked clinical 
improvement; subjects with essential hyper- 
tension showed only a minimal blood pressure 
reduction while receiving the drug. It was 
shown that after oral administration of aMPT 



45-90% is absorbed. After single oral doses 
of the drug maximum blood levels occur within 
two hours and 50% of the dose is excreted 
within 4 to 6 hours though detectable amounts 
of drug (radioactive label) are excreted for up 
to 3 days. Almost all the orally absorbed drug 
is excreted unaltered though small amounts 
appear as the decarboxylated product, amethyl- 
tyramine, and as catechol products of which 
a-methyl-dopa and a-methyl-dopamine have 
been identified. The drug appears to have defi- 
nite value in the management of cases of ma- 
lignant pheochromocytoma and in preparation 
of benign cases for surgery. In a few cases of 
the latter type the tumor has been rendered 
functionless by the drug. While a slight lower- 
ing of blood pressure is demonstrable in the 
patients with essential hypertension receiving 
large doses of the drug, it is doubtful that the 
hypotensive potency of the compound is suffi- 
cient to be therapeutically useful. Most pa- 
tients develop readily recognizable nervous 
system effects during treatment with aMPT. 
Typically the patients show sedation during 
the first two days of therapy; this may be pro- 
nounced but is usually greatly diminished with- 
in three days. At dosages of 3 gm or more per 
day, occasional patients develop tremor and 
agitation. Upon discontinuation of drug most 
patients show some degree of insomnia and 
heightened alertness and energy for several 
days before resuming their usual behavior 
pattern. Overt depression has not been ob- 
served. It is possible that aMPT would be 
therapeutically useful in other diseases such 
as thyrotoxicosis, Raynaud's disease, anxiety 
states, angina pectoris and endotoxin shock; 
further investigations are planned along these 
fines. 

The tyramine pressor test for pheochromocy- 
toma has now been evaluated in 20 patients 
with this tumor. The incidence of false nega- 
tive tests is in the range of 25-30% which is 
comparable to the incidence of false negatives 
with the histamine test. In contrast to the his- 
tamine test, however, there appears to be no 
hazard in the use of tryamine as a provoca- 
tive agent. A continuing interest in catechola- 
mine metabolism has resulted in extensive clin- 
ical experience in the medical and surgical 



118 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



management of patients with pheochromocy- 
toma. This has permitted a number of other 
studies to be performed. Contrary to data re- 
ported in the literature, no diminution in red 
cell mass or plasma volume has been found ex- 
cept in cases of severely ill patients with 
metastatic disease. In spite of the apparently 
normal blood volume it has been possible to 
obviate the typical severe hypotension follow- 
ing removal of tumor by vigorous transfusion 
with volume expanders (blood, plasma or al- 
bumin in saline solution). Experimental use of 
the /^-adrenergic blocking drug propranolol 
has shown it to be effective in the manage- 
ment of cardiac arrhythmias occurring during 
surgery. Several of the pheochromocytoma pa- 
tients studied during the past year are mem- 
bers of a single family and have exhibited the 
recently recognized triad of hyperparathyroid- 
ism, thyroid medullary carcinoma with amyloid 
deposits and bilateral adrenal pheochromocy- 
tomas. The huge increase in the work load of 
analyses of catecholamine metabolites has led 
us to set up a computer program for the calcu- 
lation of results. 

Studies on the turnover rates of radioactive 
dopamine, and norepinephrine and its metab- 
olites following the intravenous administration 
of H 3 dopa have been expanded considerably. 
The half time for turnover of dopamine, nor- 
epinephrine and vanilylmandelic acid averaged 
6 hours, 8 hours and 14 hours respectively in 
normal subjects and hypertensive patients. No 
significant difference between normals and hy- 
pertensives was noted. During treatment with 
«MPT the specific activity of norepinephrine 
was increased, a finding consistent with a 
small dopa pool. Treatment with a monoamine 
oxidase inhibitor also increased the degree of 
labelling of norepinephrine and VMA but 
slowed their rates of decay. Reserpine therapy 
greatly increased the rate of decay. The latter 
finding is one of the most convincing demon- 
strations of an effect of reserpine in clinical 
doses on catecholamine metabolism in the 
human. 

Studies of Selected Proteins 

Measurements of the urinary excretion of 
hydroxyproline (HOPr) peptides as an index 



of endogenous collagen metabolism have been 
continued in a number of clinical circum- 
stances. Hydroxyproline excretion was found 
to be normal or slightly elevated in patients 
with scleroderma. When dimethylsulf oxide 
(DMSO) was applied topically to almost the 
entire body (in doses as high as 70 ml of 90% 
DMSO) of 5 patients with sclerodema, urin- 
ary hydroxyproline was unchanged. Healing of 
ischemic ulcers was noted in 2 patients con- 
comitant with DMSO therapy. Urinary hy- 
droxyproline excretion was also found to be 
unchanged in patients with scleroderma 
receiving up to 12 gm per day of potassium 
paraaminobenzoic acid or 2 gm daily of D-peni- 
cillamine. In one patient with severe sclero- 
derma who received /?-aminopropionitrile 
(BAPN) for 4 weeks at a dose of 3 gm daily, 
urinary HOPr increased about 50%. 

During the past year a number of techniques 
have been adapted to determination of a "col- 
lagen profile" in minced specimens of dermis 
obtained by punch biopsy. This profile consists 
in part of determination of water content, to- 
tal collagen, percent of collagen extractable 
into 0.5 molar acetic acid, and the degree of 
cross-linking in soluble collagen as estimated 
by disc electrophoresis on acrylamide gel. One 
hundred twenty biopsies from patients with 
various clinical disorders in soluble collagen 
was observed along with a decrease in cross- 
linking, associated with a high water content. 
Young normal scars had a similar collagen 
profile while scars older than 6 months resem- 
bled normal skin. D-penicillamine, used in the 
treatment of Wilson's disease, cystinuria and 
(by one investigator) rheumatoid arthritis, 
was found to produce an accumulation of sol- 
uble, poorly cross-linked precursors of insolu- 
ble collagen in the skin. This is the effect seen 
in animals given lathyrogenic agents such as 
BAPN. In several patients with Wilson's dis- 
ease treated with large doses of penicillamine 
for more than 4 years, levels of soluble collagen 
were found to be 5 times those in normals (pa- 
tients =20%; normals <3.5%). Possible thera- 
peutic use of penicillamine in scleroderma is 
suggested by the finding that in this disease 
soluble collagen in dermis is consistently less 
than normal. Heritable diseases of connective 



NATIONAL HEART INSTITUTE 



119 



tissue were also evaluated. Patients with Mar- 
fan's syndrome, Ehlers-Danlos syndrome and 
osteogenesis imperfecta were found to have 
normal collagen solubility and cross-linking. 
Patients with Ehlers-Danlos syndrome were 
found to have less total collagen expressed as 
^g/HOPr/mg dry weight than normals. Sev- 
eral patients with homocystinuria — a condition 
with excess circulating and tissue levels of 
sulfhydryl-containing compounds similar to 
penicillamine — have had significantly in- 
creased solubility and decreased cross-linking 
in dermal collagen. Normal collagen profiles 
have been found in acromegaly and iatrogenic 
Cushing's disease. Adult patients with isolated 
growth hormone deficiency have less soluble 
collagen than normal. This is in contrast to 
the high levels of solubility with normal cross- 
linking in collagen of adolescents and children, 
reflecting phases of active growth and protein 
turnover. Earlier hypotheses that MarfanV 
syndrome and osteogenesis imperfecta repre 
sent abnormalities in the maturation of colla- 
gen thus were not supported by these studies. 
On the other hand penicillamine produces a 
true "collagen disorder" in man. 

The lathyrogenic agent BAPN has been ad- 
ministered carefully to five patients with 
advanced scleroderma. Maximum dose admin- 
istered was 3 gm per day and maximum dura- 
tion 4 weeks. A method was developed for 
measuring BAPN in biological materials using 
the automatic amino acid analyzer. The major 
metabolite of BAPN, cyanoacetic acid, has been 
measured by either gas or paper chromatog- 
raphy. After single or multiple oral dosage, 7- 
15% of the drug can be recovered unchanged 
in the urine, and 50-75% as cyanoacetic acid. 
The half-life of BAPN in the blood is about 
2.5 hours and no accumulation is observed on 
a 6 hour schedule of dosage. Mild allergic 
reactions developed in two of the patients, dis- 
appearing rapidly when the drug was discon- 
tinued. With the addition of the dermal colla- 
gen profile it is felt we can now proceed more 
rapidly and safely with trials of BAPN since 
the effect of the drug on collagen can be more 
accurately monitored and is thought to be the 
main basis of toxicity in experimental lathy- 
rism. The incidence of allergic reactions to the 



drug is unknown but thus far these have not 
been of sufficient severity to deter us from fur- 
ther investigations. Probably the BAPN stud- 
ies will be slowed somewhat by the recent find- 
ings that penicillamine, a current drug, also 
has powerful effects on collagen cross-linking. 
Since the cross-linking in collagen appears to 
involve the oxidative deamination of terminal 
amino groups of lysine residues, importance of 
a soluble monoamine oxidase is suggested. We 
have proceeded on the basis that plasma mono- 
amine oxidase may be the enzyme involved and 
accordingly have developed a method of radio- 
assay for this enzyme. Preliminary findings in- 
dicate that BAPN is a competitive inhibitor of 
plasma monoamine oxidase and that the mono- 
amine oxidase levels in plasma of patients re- 
ceiving pencillamine is markedly reduced. 

The conversion of radioactive proline to 
urinary peptide-bound hydroxyproline has 
been studied in young adult rats and in a sin- 
gle patient with scleroderma. The rats were 
studied several weeks after the administration 
of a single pulse label and at a time when the 
specific activity of urinary hydroxyproline 
was almost constant, reflecting metabolism of 
previously labeled and now insoluble collagen. 
When parathyroid hormone was administered 
to these rats, urinary hydroxyproline excre- 
tion increased to twice normal levels while 
hydroxyproline specific activity remained un- 
changed. This indicates that parathyroid hor- 
mone exerts its effects on the metabolism of 
insoluble collagen since the increase in urinary 
hydroxyproline originated from the labeled in- 
soluble collagen pool. Similar studies with thy- 
roid hormone indicate a mixed action, that is, 
both on synthesis and degradation. The at- 
tempt to label the collagen of a patient with 
scleroderma was successful. The specific activ- 
ity of his plasma hydroxyproline was found to 
have a half-life of about 6 days and the graph 
of his urinary hydroxyproline specific activity 
indicated contributions from at least two dif- 
ferent pools of collagen. The first had a half- 
life of 0.5 days and probably represents the sol- 
uble collagen pool while the second had a 
half-life of over 60 days, representing the in- 
soluble collagen pool. It will not be possible to 
do several studies on the effects of drugs on 



120 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



human collagen in this patient whose collagen 
pools have been labeled with C 14 in hydroxy- 
proline. 

Studies have continued on the small non- 
heme iron containing proteins which serve as 
electron carriers in low redox potential reac- 
tions in certain bacteria. These include the 
clostridial ferredoxins and a new protein 
termed "rubredoxin" which was first isolated 
and crystallized from C. pasteurianum in this 
laboratory. According to the literature ferre- 
doxin is a one electron carrier but conclusive 
evidence has now been obtained that clostri- 
dial ferredoxin is a two electron carrier. A 
search for ferredoxin-like proteins in mammal- 
ian systems has so far been unsuccessful. Stud- 
ies on rubredoxin indicate that it has a molec- 
ular weight of about 6,000, contains a single 
iron atom and has a redox potential (minus 
0.05 v) considerably higher than ferredoxin 
(minus 0.42 v). Studies on the reactivity of 
the cysteines of the molecule as well as electron 
spin resonance studies indicate that the iron 
in ferredoxin is ionically bound in contrast to 
the apparent covalent iron-sulfur bonding in 
ferredoxin. Similarity of the protein to ferri- 
chrome A, transferrin and a number of ferric 
iron chelates is suggested. Although the meta- 
bolic significance of rubredoxin is unknown it 
would appear to be useful in the study of iron 
binding mechanisms. 

Miscellaneous 

The only study in this category which has 
potential of generating a new research pro- 
gram is that concerned with oxidative phos- 
phorylation in mitochondria. The work was 
an outgrowth of attempts to extend the ferre- 
doxin studies into mammalian systems. Of sev- 
eral different findings the most interesting 
were those indicating that administration of 
large depot doses of catecholamines in rats pro- 
duces an uncoupling of oxidative phosphoryla- 
tion in heart mitochondria studied in vitro. 
This catecholamine affect cannot be shown in 
vitro using mitochondria from untreated 
animals. The time course of catecholamine ac- 
cumulation by myocardium was found to be 
similar to the P/O ratio depression produced 



following catecholamine administration. Re- 
sults following pretreatment with adrenergic 
blocking agents provided further evidence of a 
correlation between uncoupling of oxidative 
phosphorylation and elevation of myocardial 
catecholamines. Although the mechanism of the 
catecholamine effect in vivo is not yet defined, 
the phenomenon itself may contribute to our 
understanding of oxygen "wasting" that can 
be produced by catecholamines in animals and 
in man. 

LABORATORY OF TECHNICAL 
DEVELOPMENT 

Fluorescence Methods 

Methods in current use for measuring the 
absolute quantum yield of fluorescence (ratio 
of quanta emitted to number absorbed) are 
unsatisfactory. The methods are either im- 
precise or require special equipment, thus mak- 
ing it difficult to check on claims of accuracy. 
It seemed to us that the Aminco-Bowman spec- 
trophotofluorometer could be used for this pur- 
pose, since our instrument was calibrated last 
year. To demonstrate that quantum yields could 
indeed be measured with this instrument, 
studies were undetraken to measure corrected 
spectra of compounds which had been reported 
on in the literature as well as compounds 
which had never been investigated with re- 
spect to fluorescence efficiency. By publishing 
our results, it was hoped that it could be estab- 
lished that commercially available instrumen- 
tation was capable of performing a determina- 
tion hitherto thought possible only with 
specially-built components. 

The spectrophotofluorometer was evaluated 
with regard to quantum yield determinations 
by making these determinations for some 30 
solutions, for some of which there were litera- 
ture values. In general, the agreement with 
established values were good. Temperature de- 
pendence of quantum yield, and corrected 
emission spectra were determined. Sources of 
error, advantages, and disadvantages of the 
system were listed. 

Quantum yields of tyrosine, tryptophan, 
and phenylalanine were found to be about 33% 
lower than reported by Teale and Weber. Our 



NATIONAL HEART INSTITUTE 



121 



values of 0.15, 0.14, and 0.027 were recom- 
mended for use as standards in protein fluo- 
rescence work. Quantum yields of fluoro- 
phenylalanines (3 isomeric forms) were found 
to be about 0.19. 

Quantum yields and fluorescence polariza- 
tions were determined for a large number of 
dye-conjugates of bovine serum albumin. These 
are the first measurements of this type for 
such commonly used (in immunology, for ex- 
ample) dyes such as fluorescein and 1-di- 
methylaminonaphthalene-5-sulfonate. It was 
shown that dye-dye energy transfer occurs and 
causes a decrease in fluorescence yield with 
degree of labeling. Perrin plots showed that 
the depolarization was due to energy trans- 
fer in heavily labeled conjugates. Foster dis- 
tances were calculated for four types of dyes, 
and the results indicated that dipole dipole 
interaction was probable on a molecule of the 
size of BSA. 

The fluorescence quantum yield of 1-dimeth- 
ylaminonaphthalene-5-sulfonate was found to 
be lower than reported by Weber and Teale. 
In addition, the fluorescence was quenched by 
bicarbonate. It was pointed out that some 
workers had used the erroneously high figure 
of Weber and Teale in their calculations. 

Quantum yields of pyridoxamine-5-phos- 
phate, pyridoxamine, and pyridoxal were found 
to be 0.14, 0.11, and 0.048 at room temperature 
in neutral aqueous solutions. Fluorescence 
polarization spectrum of pyridoxamine-5-phos- 
phate and the temperature dependence of 
quantum yields of the three vitamin B 6 com- 
pounds were determined. The results clearly 
conflicted with a literature report that claimed 
the quantum yield of pyridoxamine-5-phos- 
phate was 0.55 under these conditions. 

While measuring the quantum yield of tyro- 
sine, it was noted that phosphate was a strong 
quencher of fluorescence. The quenching of 
tyrosine fluorescence was investigated in some 
detail and Stern-Volmer constants were de- 
termined. The tyrosine fluorescence of proteins 
was also found to be quenched by phosphate. 
Evidence from fluorescence polarization, tem- 
perature-dependence curves, the relative sizes 
of the quenching and association constants, and 
data from a larger quencher (propionate) 



showed that the quenching was mainly col- 
lisional. The use of phosphate as a probe of 
the accessibility of tyrosines in proteins was 
discussed. 

DPNH and TPNH fluorescence can be as- 
sayed in the Aminco-Bowman instrument at 
high concentrations (up to SxlO^M) by the 
use of a microcell and excitation at 395 mp. 
Under usual conditions of assay employed by 
many workers, the limit is no higher than 
10 _5 M. It was shown that non-linearity of 
the fluorescence vs. concentration curves was 
due to absorption of the incident light rather 
than absorption of the emitted light. 

A single horizontally-oriented polarizer in 
the excitation beam was found to reduce dras- 
tically ,the amount of light scatter reaching 
the detector. It was shown that this was effec- 
tive for protein emission spectral work, where 
the intense scattering of the UV excitation is 
a problem. 

To demonstrate the effectiveness of hori- 
zontally polarized excitation in spectral work 
with proteins, the emission spectra of human 
serum albumin at pH's near 4 were deter- 
mined. It was discovered that there are 
marked changes over a narrow pH range, 
which had been established by other workers 
using a variety of physical techniques to coin- 
cide with an unfolding of HSA. Analysis of 
the spectra showed that acidification was ac- 
companied by a quenching of tryptophan fluo- 
rescence and an enhancement of tyrosine 
fluorescence. This can be understood in terms 
of a decrease in tyrosine-to-tryptophan energy 
transfer. 

In addition, a small number of experiments 
on the fluorescence of dye-labeled glutamic de- 
hydrogenase and polynucleotides were per- 
formed in continuation of projects started the 
previous year. These results still must be evalu- 
ated but they indicate that the extended sensi- 
tivity we have achieved in polarization meas- 
urements may increase the usefulness of the 
spectrophotofluorometer. 

All of the material above has either been 
published or submitted for publication. 

Emission spectra of bovine serum albumin 
and the interaction of this protein with a dye- 



122 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



anilinonaphthalene-sulfonate, are under inves- 
tigation. The use of such dyes as molecular 
probes of conformational change has been pos- 
tulated by others and confirmed by us. 

Protein chemists are interested in amino 
acids labeled with 1-dimethylaminonaphtha- 
lene-5-sulfonate ("dansyl amino-acids") since 
his dye locates amino acids on chromatograms 
with about 1000-fold more sensitivity than nin- 
hydrin. The fluorescence of these derivatives 
has never been characterized, but we have pre- 
pared about 23 dansyl amino acids and puri- 
fied them by thin layer chromatography. Quan- 
tum yields and corrected spectra have been 
determined. These results will be part of a 
paper in preparation. 

Ultramicro Methods 

The helium glow photometer for the simul- 
taneous analysis of Na and K at the picomole 
(10~ 12 moles) level has been further improved 
by the addition of operating convenience and 
proven by the application to several problems 
in the analysis of renal tubule micropuncture 
samples in cooperation with the LKEM. 

The extension of the method to calcium and 
magnesium was explored and preliminary re- 
sults indicated that sensitivity was more than 
adequate being in the 10 -13 mole range. Con- 
ditions for calcium measurements have been 
worked out to eliminate interference to the 
point that accuracy in the ±2% range is con- 
firmed for calcium at the picomole level with 
good possibility of extension to 10~ 13 moles. 
The magnesium method has not been explored 
sufficiently to predict performance. 

A fluorometric method for determining nan- 
ograms of inulin obtained by micropuncture 
of renal tubules has been developed. The re- 
action of fructose, the subunit of inulin, with 
dimedone in concentrated o-phosphoric acid 
produces a moderately fluorescent product 
which has its emission peak near 400 nm and 
excitation peak near 360 mm. A concentration 
of 1 fig/ml of fructose or inulin gives a sig- 
nal that is twice the blank signal. 

The method has been shown to be more sensi- 
tive than the colorimetric method commonly 
used. However, an additional order of magni- 



tude increase in sensitivity seems desirable. We 
will attempt to improve the optical arrange- 
ment to reduce the scattered light and to re- 
duce the volume of reagent required. 

A method of producing luminescence by 
treatment with ozone has been discovered to be 
a remarkedly convenient and highly sensitive 
method of analysis of a wide variety of com- 
pounds of biological interest. Although it has 
been known for many years that ozone can 
induce luminescence in a few compounds, it has 
never been explored as an analytical method 
for biochemical application. 

Our exploration has revealed that some com- 
pounds on suitable substrates emit enough 
light on exposure to ozone gas to permit clean 
assay at the level of 10 -14 grams and a large 
proportion of fluorescent substances can be 
excited to luminescence with ozone to permit 
assay at the 10~ 12 gram level. 

Several dozen compounds have been discov- 
ered to have useful luminescence by this 
method with some indication that several com- 
pounds with relatively poor sensitivity for ul- 
traviolet fluorescence excitation have high 
sensitivity by ozone excitation. For example, 
the dye Safranine is weakly fluorescent, but 
emits strongly when excited by ozone. Ozone 
chemiluminescence methods are being com- 
pared with fluorescence methods for specific 
problems to discover what advantages the 
method has over fluorescence or other analy- 
tical techniques. 

The spectrum of emission appears to be gen- 
erally related to that of fluorescence and pro- 
vides some specificity but the low level and 
evanescent character of the emission makes 
spectral measurements tedious. 

Excitation can be produced in solution or 
dry so that chromatographic plate spots can be 
reacted for quantitative assay. The compound 
is usually consumed in the process with the 
emission of a number of photons proportional 
to the quantity present. The apparatus is very 
simple, consisting of a photometric system and 
a suitable chamber for exposing the sample to 
ozone enriched oxygen provided by passing 
tank oxygen through an electric discharge pro- 
duced by a high voltage transformer. 



NATIONAL HEART INSTITUTE 



123 



Automation of Bacteria Counts and Antibiotic 
Sensitivity Tests 

Consideration of the burden of counting 
bacteria in body fluids and the determination 
of antibiotic sensitivity by the clinical lab- 
oratory indicated a need for a simplification 
of the methodology amenable to automation 
and reduction in time required before a defini- 
tive report can be rendered. A capillary tube 
method is based on the idea that nutrient 
agar in a capillary tube will permit the growth 
and multiplication of individual organisms to 
a multiplet of organisms in less time than it 
takes to form a recognizable colony and that 
a simple scanning and memory system can be 
used to recognize and count only those or- 
ganisms that have demonstrated their viabil- 
ity by beginning to multiply. 

The capillary tubes replace not only the 
petri dishes but serve as measuring pipettes 
and provide a linear array of organisms which 
can be counted with a much simpler system 
than a planar array. 

The capillary tube is exposed to a sharply 
defined plane of illumination provided by the 
light of a neon laser. The neon laser light is 
a continuous high intensity coherent source of 
simple design available at reasonable cost that 
can be focused so fine that the resolution of the 
system can be relatively independent of the 
radial position of the organism in the capil- 
lary. 

A simple magnetic tape recording of the 
scattered light from the initial population in- 
cluding debris is recorded, the capillary tube 
incubated and a new recording indexed to 
match the previous recording is made while 
the first signal is subtracted so that only those 
organisms that have multiplied are counted. 
Variation including repeated counts and incu- 
bations as well as histograms of growth rate 
per organism can be easily obtained to in- 
crease the information obtainable. Antibiotics 
incorporated at specific dilutions in the me- 
dium can easily be assayed for more specific 
information than can be obtained by clear 
zone methods. For example, it is apparent that 
the latent period before division can be meas- 
ured for a large number of individual organ- 



isms in a short time in contrast to the tedium 
of such observations by watching single or- 
ganisms for a first sign of division under the 
microscope. 

The apparatus has been constructed and 
tested for small particles and methods devel- 
oped for eliminating noise due to defects in 
the capillary tube and light scattering from 
the agar. Resolution and sensitivity seem ade- 
quate but application to actual counts and de- 
termination of accuracy have not been com- 
pleted. 

Artificial Organs 

The membrane oxygenator developed pre- 
viously is still not in production because of 
the manufacturer's delays in getting satisfac- 
tory production methods. Some experimental 
miniature oxygenators were constructed for 
isolated organ or tumor perfusion are now un- 
dergoing experimental evaluation in the NCI 
in a cooperative project. 

The biventricular cardiac assistor develop- 
ment has progressed through some 18 modifi- 
cations to delineate the important design 
parameters. In situ tests on fibrillating as well 
as normally beating dog hearts were 
performed. Normal blood pressure and nor- 
mal pulse wave forms were achieved. Defibril- 
lation promptly restored the normal beat after 
4 hours of fibrillation in the assistor. The long- 
est duration of massage was 11 hours. A sys- 
tem to apply the assistance in synchrony with 
a normal beat has been developed to determine 
the ability of the assistor to augment the func- 
tion of a failing heart. It is presumed that 
maintenance of the integrity of the circula- 
tion will permit or accelerate healing of a 
damaged heart that would otherwise be unable 
to sustain the circulation. The assistor has been 
applied to dog hearts and the chest completely 
closed to confirm its potential for longer per- 
iods of support. 

Some improvements in the mechanical de- 
sign of the "artificial kidney" hemodialysis 
unit have improved the efficiency to permit 
high clearance at low dialysate flow. The ad- 
dition of adsorbants such as activated carbon 
further increases the effective concentration 
gradient for adsorbable substances. Most small 



124 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



molecule metabolites with the exception of 
urea are probably adsorbable. Hippuric acid 
dialysate capacity is increased 14 times. This 
represents a highly efficient compact disposa- 
ble unit not requiring - any priming blood, and 
using a minimum of dialysate. 

Fast Reaction Methods 

The developments in instruments and meth- 
ods for the study of fast chemical reactions 
in solution include the introduction of a new 
mixer, a fast pH detector system, and a new 
thermocouple amplifier. The new mixer per- 
mits 99% mixing to be achieved within 40 mi- 
croseconds or less even in the mixing of 92% 
glycerol and water. 

A small stopped flow apparatus has been 
constructed for us by the Biomedical Engi- 
neering Branch which permits the use of op- 
tical, fluorescence, pH, and thermal detectors. 
Time resolution for pH work is about 50 milli- 
seconds and sensitivity is 0.001 pH unit with 
the presently available glass electrodes. Ther- 
mal and optical time resolution is in the range 
of 2 to 5 milliseconds. 

Our instrument built by Science Products 
Corporation has improved time resolution with 
the new mixer system of 40 microseconds for 
a continuous flow thermal detector. Optical de- 
tection in stopped flow is 100 microseconds. 

Work has progressed on several fast thermal 
detectors to where a one millisecond response 
time has been achieved with glyptal coated 
copper-constantan thermocouples. Thin film 
sputtering techniques are being developed by 
Victory Engineering Corporation to coat the 
junctions with quartz in order to still further 
reduce the response time. 

The twin cell differential microcalorimeter 
has been under test for six months and is satis- 
factory for heats of reaction which produce 
30 millicalories or more in 4 ml of solution. 
The Tris-HCl reaction is being developed as 
a solution calorimetry standard and a com- 
mercial version of the instrument has been 
manufactured by Science Products Corp. 

The data restoration program developed for 
the differential calorimeter has been put into 
operation and critically tested. It has been 
shown to be able to restore electrical heater 



data from the calorimeter to better than 2 
accuracy regardless of the heat input function 
shape. Thus the adiabatic losses from the cal- 
orimeter can be corrected for, even when re- 
actions run for twenty minutes. 

Using the simulation of physical laws 
method of solving partial differential equations 
developed here, a theoretical treatment of the 
thermocouple response time measurements ob- 
tained using different thermoelectric materials 
and dimensions as well as different coating 
materials has been achieved which agrees 
within the experimental error of 2-5%. 

Chromatographic and Ultrasonic Methods 

Ultrasonic methods of measuring the content 
of sample in gas chromatography effluents de- 
veloped and applied last year have been suit- 
ably modified and tested for performance in 
liquid chromatographic effluent streams. The 
system tested measures the change in phase of 
a 20 MHz sound wave in a 1 ml cell to 0.01° 
due to a velocity change in response to a few 
micrograms of sample in 1 ml with the sensi- 
tivity limited by temperature fluctuations of 
the sample. An order of magnitude or two in- 
crease in sensitivity is available by better 
thermal stabilization. 

A variation of the method of sound velocity 
measurements has been applied to permit very 
accurate determination of sonic velocity in 
solutions. Here an interferometric technique 
based on standing wave measurements using a 
variable sound frequency which can be very 
accurately measured. The sound frequency is 
changed until the standing wave is re-estab- 
lished by accurately identifying the position of 
the standing wave peaks. Sonic velocity is com- 
puted electronically and is related to density 
and compressibility. 

As density is easily measured the method 
offers a very accurate measure of compressi- 
bility. Some preliminary measurements have 
been made to examine the possibility that 
changes in compressibility of a solution of 
large molecules would reflect the degree of 
dipole association and the influence of dielec- 
tric constant of the solvent on the association. 
The unfolding of albumin produced by low pH 



NATIONAL HEART INSTITUTE 



125 



values was disappointing but other systems 
may be more suitable. 

Miscellaneous 

A system of measuring - low gas flows (1 
to 100 ml/min) where the soap bubble meter 
is not applicable due to solubility or permea- 
tion of the soap film was developed, applied 
and published. A microperfusion pump that 
delivers 25x10"* ml/min. by programmed 
controlled thermal expansion of the oil in a 
micropipette has been developed for micro 
puncture experiments. This pump eliminates 
the need to support a heavy mechanical piston 
drive mechanism on the micromanipulator to 
provide greater freedom of motion and versa- 
tility. 

An air bearing turbine-operated grindstone 
was devised and tested to provide a very sim- 
ple method of sharpening micropuncture pi- 
pettes. 

Recovery of microgram quantities of labeled 
fatty acids on T.L.C. films without solvent ex- 
traction and the current problems of recon- 
centrating the sample was demonstrated by 
vacuum distillation in a simple apparatus. 

The utility of activated carbon for the re- 
moval of residual fatty acids bound to pure 
samples of albumin was demonstrated. 

LABORATORY OF CARDIOVASCULAR 
PHYSIOLOGY 

This report represents the last to be sub- 
mitted by the Laboratory of Cardiovascular 
Physiology of the National Heart Institute. 
Since its inception in 1954 under the direction 
of Dr. S. J. Sarnoff until its inactivation on 
July 1 of this year, the laboratory has made 
many major contributions to cardiovascular 
physiology. Besides the high quality experi- 
mental work which was considered by many 
to characterize the laboratory, the laboratory 
provided a fertile field for the training of many 
young men, many of whom subsequently went 
on to senior positions in research teaching and 
medicine. The contributions of the laboratory 
which, to a large extent, represent the contri- 
butions of Dr. S. J. Sarnoff who retired as the 
result of illness in August of last year are 



best summarized in the citation which accom- 
panied the Gairdner Foundation Award pre- 
sented to Dr. Sarnoff in 1962, . . . "in recogni- 
tion of his contribution to the knowledge of 
cardiac physiology and especially for his dem- 
onstration of the interrelated roles of the ner- 
vous system, hormones, and heart size in the 
control of cardiac performance, thus estab- 
lishing physiological principles which have as- 
sisted medical scientists to better understand 
the action of the heart in normal and diseased 
states." 

Studies on Myocardial Mechanics 

The contribution of physical and chemical 
factors to the adaptation of the heart under 
varying conditions has continued to be of sub- 
stantial interest. Several years ago this labora- 
tory clearly denned the phenomenon of homeo- 
metric autoregulation, i.e., the increase in 
myocardial contractility associated with an 
elevation of ventricular outflow resistance. It 
was shown that homeometric autoregulation 
was associated with a loss of myocardial po- 
tassium. Since it was known that decreasing 
intramyocardial K + was associated with an 
increase in myocardial contractility in isolated 
cardiac muscle, we suggested that the appear- 
ance of homeometric autoregulation was 
causally related to the potassium loss. At the 
same time the possibility existed that homeo- 
metric autoregulation was caused by physical 
changes in the heart such as shape or com- 
pliance changes. The former possibility was of 
particular interest since we had observed that 
when aortic pressure was increased isovolumic 
systolic expansion often increased. This ob- 
servation suggested the possibility that a two- 
step Frank-Starling effect may occur when 
aortic pressure is increased; the first step oc- 
curring at end-diastole and the second step 
occurring with the expansion of the circum- 
ferential fibers. We had also observed, how- 
ever, that if heart size was increased the extent 
of isovolumic systolic expansion decreases. We 
therefore undertook experiments in which, 
when aortic pressure was increased, heart size 
also increased, in order to cancel the effects 
of the two interventions on isovolumic systolic 



126 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



expansion. Such experiments showed that an 
increase in isovolumic systolic expansion was 
not necessary for the appearance of homeo- 
metric autoregulation. In these same experi- 
ments we also observed that a change in ventri- 
cular compliance occurs when aortic pressure 
is increased. The compliance change however 
could not explain the changes in myocardial 
performance. We have concluded therefore 
that while physical factors may contribute 
to the adaptation of the heart when aortic 
pressure is elevated, they do not appear to be 
necessary for homeometric autoregulation. 
With respect to the possible contribution of 
compliance changes to ventricular adaptation, 
investigations were carried out to determine if 
ventricular compliance changes during single 
or paired ventricular pacing. In the ejecting 
heart in which aortic pressure and heart rate 
were maintained relatively constant, no dis- 
cernible influence of either type of stimulation 
on the relation between left ventricular end- 
diastolic pressure and circumference was ob- 
served. In contrast, paired ventricular stimu- 
lation was observed to increase the compliance 
of the isovolumic ventricle in which developed 
tension increases during stimulation. Also, the 
increase in compliance paralleled the increase 
in developed tension. These experiments taken 
in conjunction with the experiments in which 
homeometric autoregulation was induced sug- 
gest that paired stimulation has no direct effect 
on ventricular compliance but will alter com- 
pliance if developed tension is changed sig- 
nificantly. 

An understanding of the mechanisms 
whereby the output of the heart is controlled 
is, to a large extent, an understanding of the 
factors controlling the volume of blood in the 
heart at the end of diastole and at the end of 
systole. While it is appreciated that inotropic 
interventions modify the fraction of blood 
ejected by the ventricle at each beat the role 
of physical factors in controlling stroke vol- 
ume is not always appreciated. Studies were 
carried out therefore to determine the influ- 
ence of hemodynamic factors on ejected ven- 
tricular fraction. It was observed that increas- 
ing cardiac filling increased the fraction of 
blood ejected by the ventricle while an in- 



crease in aortic pressure decreased the ejected 
fraction. These experiments show therefore that 
hemodynamic variables can modify the ejected 
fraction and point out the inadequacy of this 
measurement as an index of myocardial con- 
tractility. 

Despite the large amount of published data 
describing the phenomenon in isolated tissue, 
it was reported recently that the classical Bow- 
ditch Staircase or "Treppe" could not be dem- 
onstrated in the intact dog atrium. This find- 
ing was unexpected, at least to us, for it would 
not seem appropriate for a change in fre- 
quency of stimulation to produce the well estab- 
lished increase in ventricular contractility 
without an accompanying increase in atrial 
contractility. The problem was studied there- 
fore in this laboratory with special emphasis 
on observing the influence of heart rate on 
atrial force and rate of development of force 
over a broad range (approximately 60-200/ 
min). The results of the studies have demon- 
strated conclusively that the contractility of 
the intact dog atrium is changed with respect 
to both force and velocity when heart rate is 
modified. Also, potentiation of contraction is 
produced by extrasystoles and by continuous 
post-extrasystolic potentiation. Thus, the in- 
crease in the force of atrial contraction pro- 
duced by increasing heart rate can signifi- 
cantly augment ventricular filling during 
tachycardia. 

When heart rate was increased to high levels 
in the above study pulsus alternans was oc- 
casionally observed. Since the experimental de- 
sign was such that force gauges were usually 
placed on both the atrium and ventricle it 
was possible to observe the influence of 
alternans on both chambers. Of interest has 
been the observations of what might be 
termed asynergistic alternans. This asyner- 
gism has been observed on the same side of 
the heart between atrium and ventricle as well 
as between the right and left side of the heart. 
Of further interest is the observation that dur- 
ing alternans the strong beat occurs from a 
lower tension than the weak beat suggesting 
that pulsus alternans may be the result of 
alternating diastolic compliance. 



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127 



Myocardial Oxygen Consumption 

Studies concerning myocardial metabolism 
have been directed primarily towards de- 
termining those factors which can modify my- 
ocardial oxygen consumption. Previous studies 
using isolated cardiac muscle have been ex- 
tended to the whole heart in which it was 
found that developed tension is a major deter- 
minant of myocardial oxygen consumption. 
Studies in both the whole heart and isolated 
cardiac muscle have been the first to demon- 
strate conclusively that at a constant inotropic 
background the amount and/or rate of short- 
ening of cardiac muscle can influence oxygen 
consumption. In the working heart this in- 
fluence is small while in isolated muscle it is 
somewhat greater. When calcium is added to 
the bath containing isolated cardiac muscle 
while maintaining heart rate, preload and 
afterload constant, substantial increases in 
oxygen consumption are observed. Although 
the increase in oxygen consumption is asso- 
ciated with increases in the extent and rate of 
muscle shortening, it is also possible that a 
direct metabolic effect of the calcium is con- 
tributing to the oxygen consumption changes. 
When calcium is administered to the whole 
heart working from a high end-diastolic pres- 
sure, substantial increases in contractility 
occur without a consistent change in oxygen 
consumption. The failure to show a change in 
oxygen consumption under these conditions 
may be due to the cancelling effect of the de- 
crease in developed tension which would de- 
crease myocardial oxygen consumption and a 
direct metabolic effect which would increase 
oxygen consumption. The experiments men- 
tioned above in the whole heart would indicate 
that the extent or rate of shortening - would 
play only a small role. In any event, it is of 
interest that calcium, as we have shown also 
for acetyl strophanthidin and norepinephrine, 
can produce substantial increases in myocar- 
ial performance without a net oxygen cost. 
With respect to acetyl strophanthidin it is of 
interest that preliminary experiments suggest 
that this drug has an oxygen cost which does 
not necessarily correlate with changes in short- 
ening and/or rate of shortening. 



Influence of Drugs and Electrolytes on the 
Heart 

Previous studies from this laboratory have 
shown that the myocardial effects induced by 
various agents are associated with changes in 
myocardial potassium balance. These studies 
have been extended to determine the influence 
of calcium and sodium bicarbonate on myo- 
cardial performance and potassium balance. 
Like those of norepinephrine, the inotropic 
effects of calcium chloride have been found to 
be associated with a net gain of myocardial 
potassium and no consistent influence on myo- 
cardial oxygen consumption. The extent of the 
potassium change is directly related to the 
amount of calcium administered. The influence 
of calcium on K + balance is in contrast to the 
loss of myocardial K + associated with the ad- 
ministration of cardiac glycosides. These ex- 
periments have suggested the possibility that 
under inotropic influences the myocardial 
movements of K + and calcium occur in the 
same direction. This would then lead to the 
possibility that agents such as cardiac glyco- 
sides which cause a loss of myocardial K + 
may also produce an associated loss of myo- 
cardial calcium. 

Experiments have also been completed which 
characterize the myocardial response to so- 
dium bicarbonate infusion. This acid-base dis- 
turbance produces an initial decrease followed 
by an increase in myocardial performance. 
These changes are not dependent upon changes 
in myocardial potassium balance, coronary 
blood flow or changes in the effective catechol- 
amine background. Rather they appear to be 
the result of changes in intracellular hydro- 
gen ion concentration. It was demonstrated 
that during sodium bicarbonate infusion the 
bicarbonate ion concentration of cardiac mus- 
cle is increased. 

In view of the extensive use of Dicumarol 
and Coumadin as oral anticoagulants it was of 
interest to determine if these agents have a 
direct effect on myocardial performance when 
administered to the dog in therapeutic doses. 
Preliminary, although consistent, results indi- 
cate that over a wide dosage range Dicumarol 
depresses, while Coumadin appears to have 
little influence on the myocardium. 



128 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Studies on Electrical Stimulation of the Heart 

Previous studies have shown a minimum 
energy threshold for cardiac stimulation in 
chronic and acute preparations at pulse dura- 
tions between 0.5 and 1.0 milliseconds, using a 
constant current stimulus source. Histological 
sections of cardiac tissue from these chronic 
preparation implantation sites have shown 
>0.2 mm fibrous ring thicknesses around the 
electrode. Thus, stimulation occurs in excitable 
tissue beyond this fibrous ring, i.e., beyond 
0.2 mm from the electrode surface. 

Analysis of the electrochemical impedances 
at the electrode-electrolyte interface shows 
three components. Two of these impedances in- 
crease with time during the pulse, (activation 
and concentration impedances) while the third 
(ohmic) is constant. All vary with current 
density (not current) but only ohmic impe- 
dance is directly proportional to current den- 
sity. By studying the immediate stimulus elec- 
trode environment in electrolyte solutions and 
isolated heart tissue using glass microelec- 
trodes, it has been shown that activation and 
concentration impedances are distributed 
within 10 microns from the electrode surface. 
This is not the depolarizing region, and stimu- 
lus energy dissipated in this region does not 
contribute to stimulation. Only the ohmic im- 
pedance is distributed in the region where tis- 
sue is depolarized in chronic preparations. 
Since this impedance is directly proportional 
to the current density at the point of deploriza- 
tion, it is proposed that the electrical influence 
which leads to depolarization is also propor- 
tional to the current density in these prepara- 
tions. 

The distribution of ohmic impedance in my- 
ocardium is not uniform. It has been demon- 
strated in the isolated cat heart papillary mus- 
cle that this impedance is greater across fibers 
than along fibers. This provides a means of 
investigating "local disturbance" as a function 
of potential gradients, since for a constant 
current pulse the potential gradient is steeper 
across fibers than along fibers. 

Studies on Water and Salt Regulation 

Previous studies demonstrated a substantial 
contribution of intracardiac and intravascular 



receptors to the control of body water. The ex- 
tent to which non-cardiac vagal fibers contri- 
bute to the control of body water is not known. 
Experiments were carried out therefore to de- 
termine the influence of section of the vagal 
nerves on water diuresis in the dog in which 
cardiac vagal nerves had been ablated. In these 
animals vagal nerve section is always asso- 
ciated with a delayed antidiuresis which can 
be attributed to a decrease in free water ex- 
cretion. No consistent change in sodium excre- 
tion is seen. In some animals the antidiuresis 
is sustained while in others it is only transient. 
This variability appears to be related to the 
extent of water loading and thus the extent 
of the water diuresis. The findings suggest 
that extracardiac vagal pathways can modu- 
late free water excretion and thus possibly 
the circulating levels of ADH. At the same 
time, the failure of dogs given a heavy water 
load to show a sustained antidiuresis follow- 
ing vagotomy indicates that other controlling 
mechanisms are operating. The latter may re- 
side, at least in part, in the carotid sinus area. 

Studies on the Peripheral Circulation 

The contribution of potassium to the control 
of skeletal vasculature resistance has con- 
tinued to be of substantial interest. Previous 
studies demonstrated that the vasodilation of 
simulated muscle exercise or reactive hype- 
remia is associated with a loss of potassium 
from the muscle. When the resting muscle was 
perfused with K + so as to effect the same in- 
crease in venous K + as that which occurred 
during exercise or hyperemia, less vasodila- 
tion was observed. Studies were undertaken 
therefore to determine if changes in venous 
p0 2 can influence the vasodilator response to 
a given level of venous K + . The results of these 
experiments demonstrate that the influence 
of potassium on vascular resistance is deter- 
mined to a large extent by the oxygen supplied 
to the muscle. Thus, the lower the p0 2 of 
blood the greater the vasodilator effect of a 
given level of blood potassium. The experiments 
indicate that both oxygen and potassium con- 
tribute substantially to the control of skeletal 
muscle blood flow and that they can reinforce 



NATIONAL HEART INSTITUTE 



129 



each other in influencing vascular resistance 
in both active and resting skeletal muscle. 

The interrelationship between the vascular 
resistance changes induced in skeletal muscle 
by sympathetic stimulation and ionic balance 
has been studied. Substantial alterations in the 
total calcium concentration of the blood per- 
fusing the muscle does not influence the degree 
of vasoconstriction produced by the infusion 
of norepinephrine and epinephrine even though 
the calcium concentration was altered within 
broad extremes. The injection of CaCl 2 failed 
to increase or prolong the vasoconstriction in- 
duced by the single injection of these two 
catecholamines. It was found that these agents, 
norepinephrine and epinephrine, did substan- 
tially increase the venous p0 2 level (at con- 
stant arterial p0 2 and blood flow) when they 
produced vasoconstriction but exerted no effect 
on p0 2 when they produced vasodilation such 
as could be produced by epinephrine following 
an alpha receptor block. Of note, it was ob- 
served consistently that beta adrenergic stim- 
ulation resulted in an uptake of potassium by 
the muscle whether or not vasoconstriction or 
vasodilatation occurred. Chemically blocking 
the beta receptors prevented these potassium 
changes. Such findings indicate that these 
changes might be used in the study of adren- 
ergic receptors and could aid possibly in their 
chemical and morphological classification. 

It has been suggested by others that kinins 
act as physiological mediators of functional 
vasodilatation in glands. To test this proposal 
experiments were carried out in which the 
influence of carboxypeptidase B on the vasodi- 
latation of the cat salivary gland produced by 
stimulation of the chorda tympani nerve was 
determined. The direct infusion of kallidin into 
the artery of the salivary gland produced vaso- 
dilatation which was blocked by the simultane- 
ous administration of carboxypeptidase B. In 
contrast, Carboxypeptidase B did not block the 
vasodilatation associated with nerve stimula- 
tion. The vasodilatation was essentially blocked 
however, by a combination of atropine and a 
beta blocking agent. These experiments indicate 
therefore that the kinins play little or no role 
in the functional vasodilatation of the salivary 
gland and that the well known "atropine re- 



sistant" vasodilatation seen with nerve stimu- 
lation is the result of beta adrenergic stimula- 
tion. 

Studies on the Kallikrein-Kininogen-Kinin 
System, 

Investigations have been continued on the 
isolation and purification of the various com- 
ponents of this system. During the past year 
special emphasis was given to the isolation of 
kininogen, the substrate in plasma. Two dif- 
ferent kininogens have been isolated from 
human plasma. Both are glycoproteins con- 
taining hexose, sialic acid and glucosamine and 
have a molecular weight of approximately 50,- 
000. Although immunologically identical they 
are clearly different by several physical, clin- 
ical and biological criteria. It appears that 
kininogen I has kallidin at the C-terminal se- 
quence and kininogen II has kallidin near the 
C-terminal end of the molecule. 

LABORATORY OF KD3NEY AND 
ELECTROLYTE METABOLISM 

The research activities of the Laboratory of 
Kidney and Electrolyte Metabolism encompass 
six general fields of interest: (1) The function 
of the intact kidney as determined by micro- 
puncture analyses of luminal fluid in dogs and 
smaller animals. (2) Water and electrolyte 
transport in isolated perfused portions of the 
rabbit nephron in vitro. (3) The character- 
ization of electrolyte transport in red cells and 
the associated biochemical processes. (4) Ana- 
logous studies in epithelial membranes such as 
toad bladder. (5) Characterization of a pro- 
tein-globulin complex in mammalian plasma. 
(6) The role of renin-angiotensin-aldosterone 
system. 

Micropuncture Studies in the Kidney 

Considerable progress has been made in the 
past year concerning the nature of the counter- 
current system for urinary concentration and 
dilution. Implicit in this theory has been the 
view that an osmotic gradient develops between 
the ascending and descending limbs of the 
loops of Henle in the deeper portions of the kid- 
ney by virtue of the transport of sodium chlor- 



130 



ANNUAL REVIEW OE INTRAMURAL RESEARCH 



ide out of the ascending limb without water 
(thereby reducing the osmotic pressure) and 
the loss of water without solute by osmotic 
flow from the descending limb (thereby in- 
creasing the osmotic pressure). Although most 
workers had subscribed to this view, no direct 
supportive evidence had been available, and in 
fact a number of previous attempts limited to 
portions of the loop very close to the turn had 
failed to demonstrate any significant differ- 
ence in the osmotic pressure of the two ad- 
jacent limbs. 

In the past year advantage has been taken of 
a partial nephrectomy technique developed in 
this laboratory which permits direct visualiza- 
tion of the deeper portions of the papilla in the 
rat (see last year's report), and thereby allows 
direct micropuncture of previously inaccessible 
portions of Henle's loop. Using this preparation, 
evidence has been obtained for the presence of 
an osmotic gradient between adjacent por- 
tions of the descending and ascending limbs 
consistent with the original countercurrent 
hypothesis. Thus, the osmotic pressure of fluid 
in the thin ascending limbs of loops of Henle 
is significantly lower than that of adjacent thin 
descending limbs. Similarly, the sodium con- 
centration is lower in fluid from the ascend- 
ing limb as compared with that of descending 
limb fluid. The difference in sodium chloride 
concentration between the two limbs accounts 
for most of the difference in osmolality, and 
is consistent with the idea that active transport 
of sodium chloride out of the ascending thin 
limb is the driving force for the creation of an 
osmotic gradient in the deeper portions of the 
medulla and papilla. These findings support the 
view that the thin loops of Henle participate 
actively as countercurrent multipliers in the 
production of a hypertonic renal medulla. 

In an earlier report it had been noted on the 
basis of micropuncture sampling and analysis 
of fluid from proximal tubules in the dog, that 
antidiuretic hormone appeared to stimulate so- 
dium reabsorption in this portion of the neph- 
ron. Although these results have been reported, 
we have remained skeptical about this inter- 
pretation since an effect of antidiuretic hor- 
mone in the proximal segment on sodium trans- 
port was inconsistent with other information 



regarding its site of action. Studies were there- 
fore carried out in dogs in which samples were 
collected from the same site (the recollection 
technique) in proximal segments first during 
water diuresis and again after administration 
of antidiuretic hormone. A new remarkably 
sensitive and accurate technique for inulin de- 
termination based on fluorescence that was 
developed in the Laboratory of Technical Devel- 
opment was used. The tubular fluid/plasma- 
inulin ratio, an index of water and sodium 
chloride reabsorption in the proximal neph- 
ron, did not change significantly when diuresis 
was interrupted by the injection of vasopres- 
sin. It was concluded that the hormone has no 
effect on proximal sodium transport, and that 
the earlier conclusions were probably erron- 
eous. 

The recollection technique is to be pursued 
in a variety of studies in the future in an 
effort to determine whether the maximal so- 
dium gradient developed in the proximal tu- 
bule during administration of osmotic diuret- 
ics can be altered by administration of diuretic 
agents thought to interfere with sodium trans- 
port in this segment of the nephron. No effect 
on sodium reabsorption as determined by 
changes in the tubular fluid/plasma ratio of 
inulin had been noted in earlier studies. How- 
ever, further analysis and study are required 
before final conclusions can be reached regard- 
ing the proximal sites of action of these drugs. 

In collaboration with the Department of Med- 
icine of Duke University, an investigation of 
electrolyte transport in the distal nephron of 
the dog is also in progress. The recognition of 
the presence of distal nephrons in the superfi- 
cial cortex of the dog has heretofore been a 
difficult problem. In the past year, however, it 
has been possible to detect the presence of an 
appreciable number of distal nephrons in the 
accessible portions of the superficial cortex by 
injecting a dye (lissamine green) which makes 
it relatively easy to locate these segments of 
the neprhon. In addition to the information be- 
ing acquired concerning the normal composi- 
tion of fluid in the distal tubule, it has been 
found that the administration of furosemide 
increases the tubular fluid/plasma ratio of 
sodium in the accessible portion of the distal 



NATIONAL HEART INSTITUTE 



131 



nephron and lowers the tubular fluid/plasma 
ratio of inulin. No effect on potassium concen- 
tration has been noted. These results support 
the view that furosemide limits sodium trans- 
port in the loops of Henle and permits the 
delivery of a greater volume of less dilute urine 
to the distal convolution. 

A number of other analogous studies, in 
progress in both dogs and rats, are directed at 
characterizing transport and homeostatic 
mechanisms in individual accessible segments 
of the kidney. 

Studies of Isolated and Separated Tubules in 
Rabbits In Vitro 

In earlier reports the results of kinetic 
analyses of electrolyte transport in slices of 
renal cortex and suspensions of renal tubules 
were discussed. On the basis of these studies 
it was concluded that a minimum of two so- 
dium and two potassium compartments are 
present within tubule cells. Thought the inter- 
pretation was reasonable on the basis of the 
available evidence, it was not possible to ex- 
clude with certainty that the pools of these 
electrolytes were located in different cells or 
tubule segments. Recently a technique for di- 
rect isolation and study of individual and vi- 
able segments of rabbit tubules has been per- 
fected. Kinetic studies of isotopic exchange 
have been repeated under a variety of condi- 
tions, and it has been concluded that the exist- 
ence of several potassium compartments repre- 
sents inhomogeneity of the tubule population 
in slices of cortex and in suspensions of corti- 
cal tissue, or in other words that each nephron 
segment contains only a single potassium com- 
partment. Similar analyses with respect to Na, 
however, revealed the presence of at least two 
compartments in individual segments. 

Portions of collecting tubules have also been 
dissected freehand from rabbit kidneys and 
perfused; unidirectional and net transport of 
water and solute have been measured under 
different experimental conditions. We had pre- 
viously proposed that the permeability changes 
induced by vasopressin in toad bladder and tu- 
bule are mediated by the intracellular forma- 
tion of cyclic 3' 5' AMP. This compound pro- 



duces all the effects of antidiuretic hormone in 
toad bladder and its concentration within the 
tissue is increased following incubation with 
hormone. No direct evidence of an effect of 
antidiuretic hormone on the permeability of 
the kidney tubule to water and solute had been 
developed in the past, although all investiga- 
tors had agreed that the effect must be similar 
to that in skin and bladder. Furthermore, no 
effect of cyclic AMP on H 2 permeability in 
the kidney in vivo had been obtained. Using the 
microperf usion technique with isolated collect- 
ing tubules it has now been demonstrated for 
the first time by direct measurement that anti- 
diuretic hormone does in fact increase the per- 
meability of the tubule membrane to water in 
fashion consistent with the idea that the hor- 
mone increases either the number or size of 
aqueous channels or pores within the limiting 
membrane. It has also been found that cyclic 
3'5' AMP mimics antidiuretic hormone inso- 
far as permeability change is concerned in the 
collecting tubule, again suggesting that this 
compound may mediate the action of antidiu- 
retic hormone. No effect on urea permeability 
has been noted with either ADH or cyclic 
AMP, a finding which contrasts with observa- 
tions in toad bladder. This and other studies 
of electrolyte transport, the electrical charac- 
teristics of the membrane in proximal tubule 
and collecting tubule are in progress. 

The naturally occurring fatty acid, prosta- 
glandin (PGE), first discovered by Von Euler 
and subsequently characterized as to chemical 
composition by Bergstrom at the Karolinska 
Institute in Stockholm, appears to be a major 
component of "medullin," an extract made 
from renal medulla which lowers blood pres- 
sure in animals with reno-prival hypertension. 
Some time ago it was reported from this lab- 
oratory that PGE interfered with the permea- 
bility changes in toad bladder inuced by 
antidiuretic hormone and a physiological regula- 
tory role in the kidney was suggested. No effect 
on the changes in permeability induced with 
exogenous cyclic AMP was noted, a finding 
consistent with the view that PGE interferes 
with the mechanism by which antidiuretic hor- 
mone induces an increase in the concentration 
of cyclic AMP in the responsive tissues. PGE 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



has now been shown to inhibit the effects of 
antidiuretic hormone on the permeability of 
the collecting tubule, adding credence to the 
view that this naturally occurring fatty acid 
may be physiologically significant in renal 
tissue. 

Red Cell Studies 

Studies of the characteristics of electrolyte 
transport across red cell membranes of a va- 
riety of species have been a continuing effort 
in this laboratory. Hemolyzed preparations of 
red cells (ghosts) as well as intact red cells 
have been studied. On the basis of earlier work, 
it had been assumed that transport of ions as 
measured by unidirectional fluxes can be sep- 
arated, operationally, into at least three dis- 
tinct components; active transport, that is, 
transport against an electrochemical gradient; 
passive diffusion; and so-called exchange diffu- 
sion. Exchange diffusion is assumed to involve 
a one-for-one exchange of an ion with a coun- 
terion of the same species, that is, sodium for 
sodium or potassium for potassium. Recent 
studies have cast considerable doubt on the op- 
erational definition of exchange diffusion. Ex- 
change diffusion is by definition symmetrical, 
and any manipulation which modifies one of 
the unidirectional fluxes must exert a similar 
effect on the reverse flux. This has not turned 
out to be the case in all instances of what had 
been assumed to be exchange diffusion. Further 
evidence has developed that essentially elimi- 
nates the possibility that true exchange diffu- 
sion, sodium for sodium, or potassium for po- 
tassium, does in fact occur in the human red 
cell. It now appears that sodium-sensitive so- 
dium efflux, previously considered to represent 
exchange diffusion, and potassium-sensitive 
potassium influx both are effected by a hitherto 
underscribed component of active transport. 

In the past year progress has been made in 
further characterizing this second active pump 
system. It has been established that sodium- 
sensitive sodium efflux is, in fact, active, but 
differs from the usual form of the potassium 
and sodium active transport mechanism (Pump 
I) since it is not inhibited by digitalis glyco- 
sides, does not utilize ATP as its proximate 



energy source, does not require external potas- 
sium, and is not stimulated by an increase in 
internal sodium concentration. Active sodium 
efflux of the second type (Pump II), though 
not inhibited by ouabain alone, is inhibited by 
a combination of ethacrynic acid and ouabain 
and is sensitive to external sodium. A compo- 
nent of active potassium influx has similar 
characteristics. This transport mechanism has 
been demonstrated both in ghost preparations 
and in intact human red cells. It appears that 
under normal conditions approximately 90% of 
the sodium movement out of the red cell is by 
active transport and approximately 90% of the 
potassium entering the cell moves by active 
transport. The classical active transport process 
moves approximately 60 to 70% of the sodium 
and potassium while the newly described mech- 
anism transports the other 20-30%. It is not 
clear whether the newly discovered mechanism 
should be interpreted as requiring a second 
carrier or whether an entirely new conceptual- 
ization of the transport process is required. 

The role of the active transport in the regu- 
lation of red cell metabolism has also been 
studied in both human red cells and ghost prep- 
arations. A successful attempt has been made 
to delineate the mechanism of interaction be- 
tween cation pump activity and the control of 
glycolysis in human red cells. The flux of K 42 
and changes in enzyme activity in the glyco- 
lytic cycle were examined with and without 
ouabain and under a variety of other experi- 
mental conditions. It was observed that when 
the internal sodium of human red cells is ele- 
vated both potassium influx and lactate pro- 
duction rise, evidence of an increase in rate of 
glycolysis, and both become more sensitive to 
the inhibitory action of ouabain. This occurs 
with either glucose or adenosine as substrate. 
Fresh whole hemolysates enriched with sodium 
and magnesium will convert intermediates in 
the glycolytic chain above the triose-phosphate- 
dehydrogenase step to lactate at a rate which 
is slowed by ouabain. Metabolism of interme- 
diates below this step, that is beyond the phos- 
phoglycerate kinase step are not so affected by 
ouabain. The removal of membranes from the 
preparation eliminates the ouabain effect, evi- 
dence that the responsible enzyme is present in 



NATIONAL CANCER INSTITUTE 



133 



the membrane. Hemoglobin-free ghosts alone 
were shown to contain both triose phosphate 
dehydrogenase and phosphoglycerate kinase 
activity. The reaction rate of this two enzyme 
sequence was measured by following the con- 
version of DPN to DPNH and was found to be 
a function of the ADP concentration in the me- 
dium. On the basis of this and other studies 
it was concluded that the reaction catalyzed 
by membrane phosphoglycerate kinase is the 
step at which the sodium-potassium transport 
system influences the metabolic rate in red 
cells, and this action is possibly exerted via a 
compartmentalized ADP which is an immediate 
substrate for ghost phosphoglycerate kinase. 
The availability of the ADP is presumably de- 
pendent upon the activity of the sodium-potas- 
sium activated APTase system which is inhib- 
ited by ouabain, thereby accounting for the 
inhibition of electrolyte transport and metab- 
olism by this agent. 

Studies in Toad Bladder 

Vasopressin is known to have two character- 
istic effects on the urinary bladder of the toad: 
One, it increases the permeability to water, and 
two, it increases the rate of sodium transport. 
There is conflicting information concerning 
the energy requirement for each of these steps, 
and, in fact, as to whether metabolic energy 
was essential for the effect on permeability to 
water. As a first approach to the problem the 
effect of specific metabolic inhibitors on the 
responsiveness of the membrane to vasopressin 
was studied. The effect of these agents on the 
osmotic flow of water when stimulated by the 
hormone as well as on active sodium transport 
as estimated by short-circuit current were stud- 
ied under a variety of experimental conditions. 
It had been known that many of the metabolic 
inhibitors interfered with the active transport 
of sodium induced by vasopressin, but as im- 
plied above conflicting evidence was present 
concerning the effect of these agents on the 
permeability response insofar as water is con- 
cerned. It was observed in the present studies 
that a number of metabolic inhibitors inter- 
fered with the response of sodium transport 
to hormone and also depressed the basal rate 



of sodium transport of toad bladder. However, 
in contrast to findings of other investigators, 
a number of these agents under varying experi- 
mental conditions also interfered with the ac- 
tion of the hormone on osmotic flow of water. 
Although the effects of the individual metabolic 
inhibitors were specific, as indicated by studies 
in which changes in lactate production, CO» 
production from labelled glucose and pyruvate, 
and tissue citrate concentrations were meas- 
ured, it was not possible to pinpoint the pre- 
cise metabolic steps involved in the osmotic 
permeability response. However, it was pos- 
sible to conclude that metabolic energy is a 
necessary requirement for the alterations in- 
duced by ADH in the permeability of toad 
bladder to water. 

In addition to the above studies a major effort 
has been directed at examining the control of 
glycogenolysis in the toad bladder as changes 
are induced by anaerobiosis, vasopressin, oua- 
bain (an inhibitor of active sodium transport 
in this tissue) and the removal of sodium from 
the bathing medium. The crossover technique 
of Chance was used. In this instance, the tech- 
nique involves the measurement of the concen- 
tration of all of the glycolytic intermediates 
under control and experimental conditions. 
Rate controlling steps are detected as the site 
of reciprocal alterations in the concentration of 
substrate and product in experimental as com- 
pared to control situations. When glycogenoly- 
sis is inhibited by ouabain or the removal of 
sodium from the incubation solution, an in- 
crease in the concentration of glucoses-phos- 
phate and fructose-6-phosphate and a fall in the 
concentration of fructose diphosphate and tri- 
ose phosphate, pyruvate and lactate are ob- 
served, indicating inhibition of the conversion 
of f ructose-6-phosphate to fructose diphosphate, 
a step catalyzed by the enzyme phosphofructo- 
kinase. The opposite pattern was obtained when 
glycogenolysis was stimulated by anaerobiosis. 
In this instance the concentrations of glucose- 
6-phosphate and fructose-6-phosphate were de- 
creased, and the concentrations of fructose di- 
phosphate, triose phosphate, pyruvate and 
lactate were elevated, indicating stimulation 
of phosphofructokinase. When vasopressin was 



134 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



used to stimulate glycogenolysis, a small but 
significant rise in the concentrations of glu- 
coses-phosphate and fructose-6-phosphate and 
a further increase in the concentration of fruc- 
tose diphosphate and triose phosphate were 
observed. This is evidence in support of the 
view that activation of both phosphorylase and 
phosphofructokinase is induced by vasopressin 
under the circumstances of these studies. Ana- 
logous studies using the crossover technique 
to pinpoint alterations in the rate controlling 
enzymes in the citric acid cycle are under way 
at the present time. It is also proposed to ex- 
amine the metabolic effects induced by aldo- 
sterone in this tissue in a similar fashion. 

Physiologic studies relating the permeability 
effects of antidiuretic hormone and its ana- 
logues to the concentration of divalent cations 
in the bathing medium are also in progress. It 
has been known for some time that an increase 
in the concentration of calcium in the bathing 
medium interferes with the water permeabil- 
ity response to antidiuretic hormone but does 
not eliminate the stimulation of sodium trans- 
port. No effect was observed on the action of 
cyclic AMP on either water or sodium move- 
ment. Presumably, the inhibitory step involv- 
ing calcium precedes the formation of cyclic 
AMP within the tissue. The dissociation of the 
sodium and water permeability response to the 
hormone suggests the presence of two separate 
steps mediated by cyclic AMP, one controlling 
water movement, the other that of sodium. 
Preliminary results in a further investigation 
of this problem indicate that although the re- 
sponse to cyclic 3'5' AMP is not affected by 
high concentrations of either calcium or mag- 
nesium, the increase in the rate of flow of water 
along an osmotic gradient in bladder in re- 
sponse to a variety of hormone analogues and 
to theophylline is inhibited by both of these 
divalent cations. The response to each hormone 
analogue apparently manifests a characteris- 
tic sensitivity pattern to one or the other ca- 
tion. Further studies are in progress to delin- 
eate the effects of high concentrations of cal- 
cium and magnesium on the diffusional 
permeability of the bladder to water and urea 
with and without hormone. 



The Chemical Characterization and Physiologic 
Significance of a Cardioglobulin System 
Present in Mammalian Plasma That Increases 
The Contractility of Isolated Frog Heart 

This system is thought to contain three 
globulin components, all of which are neces- 
sary for the contractility response. In the 
past year, further purification of the factors 
has been accomplished revealing that certain 
of the components from plasma of different 
species (rat and human) differ in characteris- 
tics. This has permitted improvement of an 
assay for the presence of the cardioglobulin 
system in plasma, and it has been reestablished 
that a small increase in the fractions referred 
to as cardioglobulin A and C occurs in patients 
with long-standing hypertension, and that a 
decrease in fraction C is present in about 30% 
of patients with idiopathic myocardial failure. 
The significance of these changes is at present 
unknown. The most striking new finding, how- 
ever, has been the uniform observation of a de- 
crease in at least one of the three components 
of the cardioglobulin system in plasma of 22 
of 24 patients with lupus erythematosus. Nor- 
mal values, on the other hand, were observed 
in 17 other patients with other types of "con- 
nective tissue" disease. This aspect of the study 
will be pursued in an effort to determine the 
significance of the alterations in cardioglobu- 
lin composition in the plasma of patients with 
lupus erythematosus in the hope that it may 
yield information regarding the pathogenesis 
of the disease process. 

The Renin-Angiotensin-Aldosterone System 
and Other Humoral Factors in Homeostasis and 
Disease 

A major effort has been directed at examin- 
the renin-angiotensin-aldosterone system in 
normal, hypertensive animals and in those with 
experimentally induced heart failure and other 
causes of fluid retention. The major concern 
of this portion of the laboratory has been with 
establishing the mechanism of control of the 
secretion of aldosterone in normal and in dis- 
eased states. In addition, the nature and the 
function of certain extra-adrenal factors pro- 
moting sodium retention and excretion have 



NATIONAL HEART INSTITUTE 



135 



been investigated. Detailed background infor- 
mation concerning these problems has been 
presented in earlier reports. 

In experimental hypertension in the dog, the 
relation of plasma renin to sodium balance and 
arterial pressure was examined in a number of 
studies. The results revealed a striking corre- 
lation between plasma renin concentration and 
sodium balance, but no relationship of these 
factors to variations in arterial pressure. The 
secretion of desoxycorticosterone in experi- 
mental heart failure was studied in an attempt 
to determine if this hormone contributes sub- 
stantially to the total sodium retaining activi- 
ties in this physiologic derangement. It was 
observed that desoxycorticosterone secretion 
was normal in experimental heart failure. 
Studies were also initiated in an attempt to 
define whether or not a hepatic hormone may 
lead to renin release. Thus far the results have 
been negative. Of some significance was the 
observation that the renin-aldosterone system 
is present in lower vertebrates, such as the 
opossum and the American bullfrog. In both 
of these animals renin and ACTH increased 
aldosterone secretion and studies of the juxta- 
glomerular apparatus were interpreted as in- 
dicating hyperplasia and hypergranulation dur- 
ing sodium depletion. 

Evidence has been obtained in the past in 
this and other laboratories, on the basis of 
cross-circulation studies, that a humoral fac- 
tor is involved in the natriuresis of saline load- 
ing. If one infuses saline into a donor animal 
and cross-circulates its blood into a recipient, 
a significant augmentation in sodium excre- 
tion occurs in the recipient animal. Attempts 
to determine the locus of secretion of this un- 
known humoral factor by cross-circulation of 
blood from hepatectomized, decapitated or ne- 
phrectomized dogs have thus far yielded nega- 
ive results. Further studies, however, are in 
progress. Attempts have also been made to ob- 
tain information concerning the afferent input 
into the system involved in the natriuresis of 
saline loading. The natriuretic response to the 
infusion of blood was compared to the response 
to infusion of saline. It was noted that blood 
as well as saline produced substantial increases 
in sodium excretion. Additional studies, how- 



ever, are necessary to define adequately those 
factors which lead to certin differences in the 
responses to blood and saline which were ob- 
served in these studies and to determine 
whether or not the afferent input for the natri- 
uretic mechanism is related mainly to volume 
expansion or to other factors as well. 

LABORATORY OF BIOCHEMISTRY 

Section on Enzymes 

The Section on Enzymes continues to direct 
its attention to the following major areas of in- 
vestigation: (1) the intracellular regulation of 
enzyme activities, with particular emphasis on 
the regulation of divergent metabolic path- 
ways; (2) the uptake of amino acids by iso- 
lated cytoplasmic membrane preparations; 
(3) the metabolism of amino acids; (4) the 
metabolism of one-carbon compounds; (5) 
cystathionine synthesis and trans-sulfura- 
tion; (6) the mechanism of action of vitamin 
B 12 , and; (7) the dissimilation of heterocyclic 
compounds. These studies embrace broad areas 
of enzyme function in metabolism and have 
led inadvertently to the discovery of several 
biochemical processes in which vitamin B 12 
derivatives and non-heme iron electron carrier 
proteins (ferredoxins) are of fundamental 
significance. These special processes have there- 
fore been the target of intensive study since 
they represent model systems whose investi- 
gation may help to elucidate the mechanism 
of action of vitamin B 12 compounds and non- 
heme iron proteins in intermediary metabolism. 

The Regulation of Divergent Biosynthetic 
Pathways of Metabolism 

Cumulative Feed-back Inhibition of Glu- 
tamine Synthetase. — Previous studies in this 
laboratory have shown that the glutamine syn- 
thetase of Escherichia coli is subject to 
feedback inhibition by eight different end-prod- 
ucts of glutamine metabolism; namely, tryp- 
tophan, histidine, AMP, CTP, carbamyl-P, 
glucosamine-6-P, glycine and alanine. On the 
basis of kinetic studies it is concluded that 
each of these compounds is independent in its 
action. When tested individually at saturating 



136 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



concentrations each product will cause only 
partial inhibition of the glutamine synthetase 
activity; however, these effects are cumulative, 
and when all eight compounds are present si- 
multaneously, over 95% of the enzyme activ- 
ity is inhibited. 

In efforts to understand the mechanism of 
this unique cumulative feed-back inhibition 
pattern, the glutamine synthetase of E. coli 
has been isolated as a homeogeneous, crystalline 
protein and some of its properties have been 
determined. 

On the basis of various physical and chemi- 
cal measurements including sedimentation 
analysis, light scattering measurements, disc 
gel electrophoresis, amino acid analysis and 
fingerprint analysis of tryptic digests, it has 
been established that the enzyme has a molec- 
ular weight of about 680,000 and that it is 
composed of 12-14 probably identical subunits. 
Complete disaggregation of the enzyme into 
its catalytically inactive subunits (MW = 49,- 
000-53,000) is achieved by treatment either 
with 4 M guanidine or with 1.0 M urea plus 
0.01 M EDTA. After complete disaggregation 
by the latter method (4°, pH 7.0), the addi- 
tion of 0.02 M MnCl 2 causes reaggregation of 
the subunits to form an oligomer that is in- 
distinguishable from the native enzyme with 
regard to sedimentation constant and disc gel 
electrophoretic mobility. This reaggregation is 
accompanied by restoration of up to 80% of 
the normal catalytic activity. 

The enzyme contains 4 cysteine residues per 
53,000 molecular weight subunit. At high pro- 
tein concentrations, 300 /xg/ml, these sulfhy- 
dryl groups are all inaccessible to titration 
with alkylating agents or with Ellman's rea- 
gent. However, quantitative titration of the 
sulfhydryl groups with Ellman's reagent is 
possible following denaturation with dodecyl 
sulfate or 4 M guanidine. On the other hand, 
in dilute solutions, 250 ju.g/ml, the enzyme 
does react slowly with alkylating agents such 
as p-chlor-mercuriphenyl sulfate (PCMPS) in 
the absence of protein denaturants. This alky- 
lation is attended by dissociation into catalyti- 
cally inactive subunits of undetermined size. 

From considerations of the kinetics of gluta- 
mine synthetase inhibition and the diverse 



structures of the 8 different end-products of 
glutamine metabolism it was previously con- 
cluded that the enzyme (for that matter prob- 
ably each of the identical subunits) possesses 
separate binding sites for each of the 8 feed- 
back inhibitors. Efforts to achieve differential 
inactivation of these separate binding sites by 
means of selective denaturation through con- 
trolled proteolytic digestion were unsuccess- 
ful. On the other hand, the existence of sepa- 
rate binding sites for each inhibitor is 
supported by investigations of the kinetics of 
inhibition and also from studies on the effects 
of the various feed-back inhibitors on the in- 
activation of the enzyme by PCMPS. For 
example, the inhibition of glutamine synthe- 
tase by tryptophan, glycine and CTP is com- 
petitive with respect to the substrate gluta- 
mate, yet these three inhibitors apparently do 
not react at a common binding site since tryp- 
tophan protects the enzyme from inactivation 
by PCMPS, whereas CTP enhances the inac- 
tivation and glycine has no effect. It is evi- 
dent that histidine and glucosamine-6-P in- 
volve binding sites that are different from 
those that react with the other inhibitors since 
these two compounds are competitive only with 
respect to the substrate ammonia; yet histi- 
dine protects the enzyme from PCMPS inacti- 
vation, whereas glucosamine-6-P has little 
effect. Finally, the three end-products that are 
noncompetitive inhibitors of glutamine syn- 
thetase activity are differential in their effects 
on PCMPS inactivation. Thus, AMP protects 
against PCMPS inactivation, carbamyl-P en- 
hances the inactivation and alanine has little 
effect. These results are most compatible with 
the conclusion that the enzyme possesses sepa- 
rate binding sites for each of the 8 feed-back 
inhibitors. 

In order to determine the number of bind- 
ing sites for tryptophan, some preliminary 
studies have been carried out in collaboration 
with Dr. Gregorio Weber at the University of 
Illinois. For these studies advantage was taken 
of the fact that the tryptophan analog, 6-nitro- 
tryptophan (NT), will replace tryptophan as 
a feed-back inhibitor and protects the enzyme 
against PCMPS inactivation. Since NT is non- 



NATIONAL HEART INSTITUTE 



137 



fluorescent and possesses a strong light absorp- 
tion band above 300 m^, its binding to the 
enzyme is accompanied by a quenching of pro- 
tein fluorescence at 335 m/i. By measuring the 
degree of quenching as a function of NT con- 
centration, it has been estimated that there 
are about 33 moles of NT bound per mole of 
enzyme at saturation concentrations, and that 
the dissociation constant is 5x1 -5 M. From 
these data it is concluded that between 2 and 
3 equivalents of NT are bound to each subunit. 
The further observation that the quenching 
of protein fluorescence by NT is not influenced 
by saturating concentrations of alanine, gly- 
cine or glutamate, support the conclusion that 
the various feedback inhibitors are independ- 
ent in their actions and that the binding of 
one inhibitor is not influenced by the binding 



of another. If the results of NT binding using 
the fluorescence technique can be confirmed by 
the more conventional method (i.e., by equi- 
librium dialysis) this fluorescence technique 
will be exploited further to study the interac- 
tions of the enzyme and the various end- 
products and substrates. 

Finally, a new highly sensitive method has 
been disclosed which permits more careful 
kinetic measurements of glutamine synthetase 
activity. In this method the ADP formed as 
a by-product of the glutamine reaction (1) is 
coupled with reactions catalyzed by pyruvate 
kinase (reaction 2) and lactate dehydrogenase 
(reaction 3). The oxidation of DPNH in the 
over-all reaction 4 is followed spectrophoto- 
metrically and under appropriate conditions is 
a measure of the glutamine synthetase activity. 



ATP + NH 3 + glutamate — > glutamine + Pi + ADP 
ADP + p-enolpyruvate — >ATP + Pyruvate 



(1) 

(2) 



Pyruvate + DPNH + H + — >DPN + lactate 

Sum: Glutamate + NH 3 + DPNH + p-enolpyruvate 

+ 
+ H+ — > glutamine + lactate + DPN 



(3) 

(4) 



Using this method it was discovered that 
glutamine synthesis is not a linear function of 
time but exhibits a short lag of 1-2 minutes 
before maximum velocity is reached. The lag 
can be eliminated if this enzyme is given a 
prior incubation with very high concentrations 
of glutamate at room temperature. The concen- 
tration of glutamate (0.4 M) required for this 
effect is considerably greater than that needed 
to saturate the enzyme as a substrate in gluta- 
mine synthesis. The results suggest that gluta- 
mate at high concentration induces a confor- 
mational change in the enzyme to a form that 
is catalytically more active. Removal of the 
glutamate by sephadex filtration results in the 
immediate return to a preparation that exhib- 
its a lag phase. 

In order to determine the generality of the 
cumulative feed-back control mechanism in the 
regulation of glutamine synthetase, the inhibi- 
tion patterns of the enzyme from various 
sources have been examined. Ten different mi- 
croorganisms were studied including 5 aerobic 



bacteria representing 4 different generic types, 
and one species each of an obligately anaerobic 
organism, a photosynthetic bacterium, a mold, 
a yeast and a green alga. Although significant 
differences were found in the regulation pat- 
terns with respect to the nature and extent 
in inhibition, in all instances the glutamine 
synthetase activity was subject to inhibition 
by several or all of the end-products of gluta- 
mine metabolism. In most cases, a cumulative 
feedback mechanism seems to operate for 
majority of the inhibitors; however, in some 
instances certain pairs of inhibitors are antag- 
onistic or synergestic in their action. Of spe- 
cial interest is the strongly synergistic effects 
of AMP and glutamine or of AMP and histi- 
dine that are observed with the glutamine 
synthetases of Bacillus cereus and Bascillus 
Licheniformis. The enzymes from these organ- 
isms promise to be of unique interest from the 
standpoint of allosteric interactions and puri- 
fication of the enzyme from B. licheniformis 
is in progress. 



138 



ANNUAL REVIEW OE INTRAMURAL RESEARCH 



The Uptake of Amino Acids by Isolated 
Cytoplasmic Membrane Preparations 

In an effort to determine the mechanism of 
carrier-mediated transport, the uptake of 
amino acids and sugars by isolated cytoplasmic 
membranes from Escherichia coli is under in- 
vestigation. The membranes are prepared from 
penicillin-induced spheroplasts by osmotic 
shock in hypotonic salt solution containing 
DNase, followed by the addition of EDTA, and 
are separated from trace amounts of intact 
cells and spheroplasts by sucrose density gra- 
dient sedimentation. Homogeneity of the prep- 
arations was established by electron and phase 
contrast microscopy, viability assays, chemi- 
cal and enzymatic assays, and disc gel electro- 
phoresis. The isolated membranes contain less 
than 5.0% of the DNA or RNA and less than 
1% of the soluble cytoplasmic protein of the 
cell. 

Previous studies showed that such mem- 
brane preparations from wild-type E. coli W 
catalyze the energy dependent uptake of gly- 
cine, whereas similar preparations from a mu- 
tant strain (WS), having a defective glycine 
uptake capacity, failed to do so. Although 
these results indicated the glycine uptake by 
the membranes is the manifestation of a 
physiologically significant transport process, 
interpretation of the results was complicated 
by the discovery that the glycine taken up by 
the membranes was largely converted to phos- 
phatidyl ethanolamine, probably via serine and 
phosphatidyl serine. As a consequence, the in- 
tramembranal concentration of free glycine 
did not significantly exceed the external con- 
centration. It was therefore not possible to 
determine if the energy requirement for gly- 
cine uptake is concerned solely with a glycine 
specific carrier mediated concentration system 
or if it is related also to the conversion of gly- 
cine to phospholipids. 

In view of these considerations attention was 
turned to the uptake of proline whose poten- 
tial metabolic fate is more restricted. It has 
been found that membrane preparations from 
E. coli W6, a proline auxotroph, catalyze the 
energy dependent concentrative uptake of pro- 
line; whereas comparable preparations from a 



mutant strain E. coli W157, that is deficient 
in proline uptake and exchange capacity, do 
not catalyze concentrative uptake of proline. In 
contrast to the results with glycine, the mem- 
branes from the parental strain (W6) estab- 
lish an intramembranal concentration of pro- 
line strain (W6) establish an intramembranal 
concentration of proline that is 50 times 
greater than the external concentration. More- 
over, more than 80% of the proline taken up 
can be accounted for as free proline. 

Proline uptake by W6 membranes exhibits 
saturation kinetics at low external proline 
concentrations, is oxygen dependent and is 
stimulated by glucose; it is inhibited by DNP, 
iodoacetamide, amytal and by hydroxyproline 
but not by other amino acids or by Chloromy- 
cetin, or KCN. Fixed proline exchanges with 
external proline and hydroxyproline but not 
with other amino acids. Proline uptake by 
W157 membranes exhibits linear kinetics over 
a 100,000-fold concentration range and is not 
stimulated by glucose; it is not inhibited by 
DNP, iodoacetamide, or hydroxyproline in 
concentrations sufficient to produce maximum 
inhibition of proline uptake by W6 mem- 
branes. Proline taken up by W157 membranes 
does not exchange with external amino acids. 
When membranes are allowed to accumulate 
proline, sonicated or solublized with detergent 
and then passed over a Sephadex G-50 column, 
less than 5% of the radioactivity is associated 
with the large molecular weight components 
coming off the column at the front. This find- 
ing indicates that the intramembranal proline 
is not bound to a membrane component by a 
covalent bond. 

Finally, preliminary studies on thiomethyl- 
galactoside uptake by membrane preparations 
from constitutive and deletion mutants for 
the lactose operon have yielded results thus far 
similar to those reported above for the proline 
system. 

These results indicate that membrane prep- 
arations provide an excellent medium for stud- 
ies on the mechanism of carrier mediated 
transport. In future studies efforts will be 
made to determine the nature of the energy 
dependent process and to establish the mech- 
anism of permease action. 






NATIONAL HEART INSTITUTE 



139 



The Metabolism of Amino Acids 

Reductive Deamination. — Studies on the 
mechanism of the anaerobic phosphorylation 
associated with electron transfer during the 
reductive deamination of glycine by soluble 
enzyme preparations of Clostridium sticklandii 
have been continued. As noted earlier, the 
glycine reductase system is extremely complex, 
it has been resolved into at least eight separate 
protein components some or all of which are 
required for glycine reduction depending upon 
the nature of the electron donor system em- 
ployed. Two of the protein fractions, Fraction 
GR and Protein A are always needed irrespec- 
tive of the electron donor. When TPNH or 
DPNH are electron donors the respective TPNH 
or DPNH flavoprotein dehydrogenase are also 
required and in addition ferredoxin (the non- 
heme iron electron carrier protein), and a pro- 
tein fraction B, (obtained by precipitation 
between 0.55 and 0.9 saturated ammonium sul- 
fate), an arsenite sensitive protein fraction 
and an acetyl CoA dependent enzyme compon- 
ent are all needed. All of these protein com- 
ponents, with the possible exception of ferre- 
doxin and DPNH dehydrogenase, which have 
not been tested, are required also when re- 
duced methyl viologen is the electron donor. 
On the other hand, when a dimercaptan such 
as dimercaptothreitol is the electron donor only 
Fractions GR and Protein A and possibly the 
arsenite sensitive catalyst and the acetyl CoA 
dependent enzymes are needed. Thus, the arti- 
ficial dimercaptan electron donors appear to 
bypass a highly complex electron transport 
system needed in the normal metabolism. 

The partial purification of Protein A, an aci- 
dic low molecular weight protein component 
of the glycine reductase system, was described 
previously. Further studies show that this is 
a sulfhydryl protein that is inactivated by 
alkylation of its reduced form with iodoaceta- 
mide. The aggregated, less acidic, partially in- 
active form of protein A can be disaggre- 
gated and reactivated, to some extent, by 
prolonged treatment with dimercaptothreitol in 
3 M guanidine. A possible phosphoryl group 
carrier function for protein A was suggested 
by the fact that orthophosphate partially pro- 



tects protein A from inactivation and aggre- 
gation. However, in crude extracts no phos- 
phorylation of protein A with P :t2 -labeled 
ATP or acetyl-P could be detected. On the 
other hand, it may be highly significant that 
under these conditions P 32 is readily incor- 
porated into the flavin nucleotide prosthetic 
groups of the TPNH and DPNH flavoprotein 
dehydrogenases whose isolation was previously 
reported. 

Lysine Fermentation. — The fermentation 
of lysine by cell-free extracts of C. sticklandii 
and Clostridium M-E leads to the formation 
of ammonia, acetate, butyrate and ATP ac- 
cording to the over-all equation: 

Lysine+2H.20 + Pi+ADP ATP + 2NH 3 + 
acetate + butyrate 

Previous studies have shown this to be a com- 
plicated process requiring the participation of 
several enzymes, and a number of cofactors in- 
cluding CoA, DPN, Fe ++ , «-ketoglutarate, pyr- 
uvate, acetyl CoA and vitamin B 12 coenzyme. 

From the standpoint of intermediary me- 
tabolism, the identification of /3-lysine and 3,5- 
diaminohexanoic acid as early intermediates 
in lysine degradation discloses a very unusual 
biochemical mechanism. The conversion of 
a-lysine to /3-lysine involves migration of the 
a-amino group to the /3-position. This reac- 
tion was first described by Barker et al. and 
has now been shown to be the first detectable 
step in the fermentation of lysine by Clos- 
tridium sticklandii and Clostridium-ME. The 
reaction is sensitive to sulfhydryl reagents and 
probably involves a cofactor since it is inhib- 
ited by treatment of enzyme preparation with 
either charcoal or Dowex-1-formate. 

The second detectable step is the conversion 
of /3-lysine to 3,5-diaminohexanoic acid. This 
unusual reaction involves migration of the 
amino group from the 6 to the 5 carbon atom. 
From the biochemical point of view this trans- 
formation is of special interest since it in- 
volves participation of vitamin B 12 coenzyme; 
in addition, ATP, Mg ++ and probably pyruvate 
are needed. 

When a-lysine or /?-lysine are fermented by 
cell-free extracts at pH 7.0 (one pH unit be- 
low the optimum for the over-all fermenta- 



140 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tion), 3,5-diaminohexanoic acid accumulates 
in substantial amounts (30%) and has been 
isolated in good yield by chromatography 
on Dowex 50 and silicic acid columns. The 
enzymatic product was identified as 3,5- 
diaminohexanoic acid or the basis of the infra 
red and NMR spectra and by high resolution 
mass spectrometry of the enzymatic product 
and of its 8-lactam and N,N-dibenzoyl deriva- 
tives. Authentic samples of 3,5-diaminohexa- 
noic acid and its S-lactam derivative were pre- 
pared by reaction of ammonia with sorbic acid 
under high pressure. The synthetic products 
were shown to be identical with the enzymatic 
products by comparison of their chromatog- 
raphic behaviors and their NMR and mass 
spectra and of their abilities to be fermented 
to acetate, butyrate and ammonia by cell-free 
extracts of C. sticklandii. 

Future studies will be aimed at the purifica- 
tion of the enzymes involved in the synthesis 
of /Mysine and 3,5-diaminohexanoic acid. In 
particular the role of vitamin B ]2 in the con- 
version of /3-lysine to 3,5-diaminohexanoic 
acid will be studied in detail. 

One Carbon Metabolism 

Methane Fermentation. — A role of a 
methyl-B 12 derivative in the enzymatic synthe- 
sis of methane is suggested by the previous 
studies in this laboratory showing that crude 
extracts of Methanosarcina barkeri catalyze 
the synthesis of methyl-B 12 from methanol 
and vitamin B 12s , and in the presence of ap- 
propriate electron donors, they catalyze also 
the conversion of methyl-Bi 2 to methane. It 
has now been established that formaldehyde, 
the carboxyl group of pyruvate and unidenti- 
fied endogenous substrates present in crude 
extracts are also able to react enzymatically 
with B 1M to form methyl-B 12 . 

In order to elucidate the mechanism of 
methyl-Bi 2 synthesis, the enzymatic reac- 
tion between methanol and B 12s has been 
studied in detail. Methyl-B, 2 synthesis is stim- 
ulated by a reducing atmosphere (H 2 vs 
argon) and is inhibited by aminopterin, ar- 
senite, intrinsic factor and o-phenanthroline; 



all of which inhibit also the conversion of 
methanol to methane. 

The enzyme system that catalyzes the syn- 
thesis of methyl-B 12 has been resolved by 
DEAE-cellulose chromatography into three 
fractions, protein fractions R and A, and a frac- 
tion containing a heat stable cofactor, all of 
which are necessary in addition to ATP and 
Mg ++ in order to obtain methyl-B 12 synthe- 
sis. The active component of fraction R is 
probably a highly acidic red protein that has 
a spectrum typical of a B 12 -protein. This com- 
ponent is enriched throughout a 30-50 fold 
purification of the active protein by means of 
chromatography on Bio-gel P-100 or P-200 and 
TEAE-cellulose or DEAE-Sephadex. Fraction 
A contains at least two components which are 
required for optimal methyl-B 12 synthesis. One 
component has been identified as a ferredoxin- 
type protein by its spectrum in the oxidized 
and reduced states, and also by the fact that 
it will replace ferredoxin in the DPN-linked 
hydrogenase system of Clostridium kluyveri. 
Whether or not the ferredoxin-like activity of 
fraction A is identical with clostridial ferre- 
doxins remains to be established. The function 
of the other protein component in fraction A 
that is required for methyl-B 12 synthesis is not 
known. 

The heat stable cofactor obtained in the 
effluent fraction from DEAE-cellulose columns 
has not yet been identified, but it has been 
partially purified and some of its properties 
have been determined. It is stable in 2 N HC1 
for 2 hours at 80° or in 0.1 N NaOH for 30 
minutes at 100° C. It is anionic as shown by 
elution from Dowex-1-Cl with 0.1 N HC1. It 
is quantitatively adsorbed on charcoal and 
eluted with ammoniacal ethanol. The most 
highly purified preparations have no absorp- 
tion in the visible spectral region, but do ab- 
sorb at 260 m/x; it remains to be seen if the 
absorption is due to the active component. 

It has not been possible to resolve the en- 
yzme system catalyzing the over-all conversion 
of methanol to methane into its component 
parts, presumably because of the oxygen la- 
bility of the system. Nevertheless, when low 
concentrations of crude extract are used, sig- 



NATIONAL HEART INSTITUTE 



141 



nificant stimulation of the over-all conversion 
of methanol to methane is obtained by adding 
ATP, CoA and each of the resolved compon- 
ents (i.e., protein Fractions A and R and the 
heat stable cofactor) shown to be required for 
the synthesis of methyl-B 12 . This is further 
evidence that the enzyme system catalyzing 
the synthesis of methyl-B 12 is involved in the 
synthesis of methane. In addition to the puri- 
fied heat stable cofactor described above, the 
conversion of methanol to methane appears to 
require still another heat stable cofactor pres- 
ent in boiled cell extracts. 

Further studies will be made to characterize 
the heat stable cofactor and to establish the 
interrelationship of the various protein frac- 
tions involved in methyl-B 12 synthesis and in 
the synthesis of methane from methanol. 

Synthesis of Acetate from C0 2 . — Previous 
studies in this laboratory have shown that cell 
free extracts of Clostridium thermoaceticum 
catalyze the total synthesis of acetate from 
C0 2 . A role of a methyl-cobalamin derivative 
in this synthesis is indicated by the fact that 
the incorporation of C0 2 into acetate is par- 
tially inhibited by intrinsic factor and by the 
fact that cell-free extracts catalyze the de novo 
synthesis of Co-methyl-cobalamin from C0 2 , 
B 12s and pyruvate, and are able to utilize the 
methyl group of Co-methyl-cobalamin for the 
synthesis of the acetate methyl group. 

As reported last year the enzyme system 
that catalyzes the latter reaction has been re- 
solved into two protein fractions which to- 
gether with clostridial ferredoxin from other 
sources are required for the utilization of Co- 
methyl-cobalamin in acetate synthesis. In con- 
tinuing studies a third protein fraction has 
been obtained from crude extracts that will re- 
place the ferredoxin requirement. This fraction 
possesses ferredoxin-like activity since it will 
substitute for ferredoxin in the hydrogenase 
system of C. kluyveri; however, it has an atyp- 
ical chromatographic behavior on DEAE-cellu- 
lose columns that differentiate it from other 
ferredoxins. The possible relation of this ferre- 
doxin-like compound to the recently reported 
favodoxin is under investigation. 

In addition to the three protein fractions, 
the utilization of Co-methyl-cobalamin for 



acetate synthesis requires the presence of 
pyruvate and CoA; other «-ketoacids except 
a-ketobutyrate are not able to replace pyru- 
vate. The specific role of pyruvate and CoA 
could be (1) to serve as a source of acetyl-CoA 
ior acetyl-P; (2) to serve as a source of low 
potential electrons, as for example reduced fer- 
redoxin; or (3) to serve as a source of an 
active carboxyl group via a transcarboxyla- 
tion mechanism. Thus far attempts to demon- 
strate one or more of these functions have 
failed. The pyruvate requirement cannot be re- 
placed by acetyl-P or acetyl-CoA or systems 
generating these compounds; nor can it be re- 
placed by other sources of low potential elec- 
trons such as reduced methyl viologen nor by 
reduced ferredoxin generated by the hydro- 
genase system of C. kluyveri; moreover, a bio- 
tin dependent transcarboxylation function is 
contraindicated by the insensitivity to avidin 
of the over-all acetate synthesis or of the ex- 
change of C0 2 with the pyruvate carboxyl 
group, that is also catalyzed by cell-free ex- 
tracts. 

In future studies further efforts will be made 
to identify the individual steps in the metab- 
olism of Co-methyl-cobalamin and especially 
to establish the roles of pyruvate or of ferre- 
doxin in the synthesis of acetate. 

Cystathionine Biosynthesis and Trans- 
sulfuration 

The major findings of the past year are in 4 
areas. The first concerns the reactions mediat- 
ing cystathionine synthesis from cysteine and 
homoserine (biosynthetic trans-sulfuration) in 
fungi. Two decades have elapsed since this 
process was first postulated to occur, and since 
3 genes were defined which appeared to govern 
it in Neurospora. Mutation in one of these 
genes has now been found to result in: (a) 
failure to accumulate the acetyl ester of homo- 
serine; (6) ability to respond to this ester 
nutritionally; and (c) lack of an enzyme cat- 
lyzing an exchange between the ester and free 
homoserine. 

Second, one of the enzymes of bacterial bio- 
synthetic transsulfuration, cystathionine y- 



142 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



synthetase, has been obtained in pure form 
from Salmonella, and its physical properties 
and subunit structure have been characterized. 
Third, the cystathionine y-cleavage enzyme of 
Neurospora (reverse trans-sulfuration) has 
been found to be derepressed 60-fold by sulfur 
deprivation. Such striking derepression in this 
organism has previously been associated with 
enzymes that appear spontaneously only dur- 
ing sexual differentiation. However, in con- 
trast to the latter enzymes, cystathionine 
y-synthetase was not elevated after total star- 
vation. A non-sporulating yeast showed no re- 
sponse to sulfur deprivation. 

A unique kind of pyridoxal phosphate ca- 
talysis has been detected through the effect 
of added N-ethylmaleimide on y-elimination 
reactions. Instead of a-ketobutyrate, a new 
product is formed, identified last year as struc- 
ture I. 

CH 3 

I 
CHCOCOOH 







R 



The structure has been confirmed by mass spec- 
troscopy. The major findings of the past year 
were concerned with the mechanism of for- 
mation of compound I. The discovery that 
/^-substituted 4-carbon amino acids also react 
to yield I has restricted the possible points of 
maleimide attack to intermediates in enamine- 
ketimine tautomerization. The possibility that 
free aminocrotonate was the reactive interme- 
diate was emphasized by the discovery of a 
slow non-enzymic formation of compound I 
from a-ketobutyrate and maleimide, which 
specifically required ammonia as catalyst. Re- 
cent work has been concerned with the latter 
reaction. A comparison of the effects of am- 
monia and of a series of amines on the rate of 
solvent exchange with the ^-hydrogen of 
a-ketobutyrate has made it unlikely that the 
non-enzymic reaction involves the formation of 



a-aminocrotonate. A possible clue to the cat- 
alytic action of ammonia has come from the 
finding that the rate of hydrolysis of malei- 
mides is decreased in its presence. 

The Mechanism of Action of Vitamin B lt 
Coenzymes 

Ethanolamine Diaminase. — It was pre- 
viously established in this laboratory that the 
conversion of ethanolamine to acetaldehyde 
and ammonia by a clostridial enzyme is a 
vitamin B 12 coenzyme dependent reaction. Pur- 
ification of the ethanolamine deaminase was 
therefore undertaken in order to obtain a sim- 
ple model system that could be used for inves- 
tigation of the mechanism of cobamide 
coenzyme action. 

A relatively simple three step procedure has 
been developed for the isolation of the enzyme 
from cell-free extracts of ethanolamine adapted 
cells. The isolated deaminase is homogeneous 
as judged by its sedimentation in the ultra- 
centrifuge and its mobility on disc gel electro- 
phoresis. The pure enzyme has an absolute 
requirement for cobamide coenzyme and for 
potassium ions. The pH optimum is from 6.8 
to 8.2. Preincubation of the deaminase and 
coenzyme in the absence of substrate results 
in diminished activity within a few seconds 
and over 90% inactivation within five minutes. 
At low concentrations of the cobamide coen- 
zyme there is a significant lag period in the 
deamination reaction which decreases with in- 
creases in coenzyme concentration. 

The molecular weight of the deaminase is 
approximately 500,000 gm. as determined by 
the Yphantis sedimentation equilibrium 
method. The sedimentation coefficient is 14.5 
Svedbergs. Preliminary studies indicate that 
4 M guanidine causes the enzyme to dissociate 
into subunits of about 55,000 gm. molecular 
weight. The enzyme is stable indefinitely in 
liquid nitrogen and for several days at 4° C 
in concentrated solution. Amino acid analysis 
shows 3 half cystine residues per minimum 
molecular weight of 28,660 gm. Parachloro- 
mercuriphenylsulfonate is the only sulfhydryl 
reagent of several tested that inhibited the 



NATIONAL HEART INSTITUTE 



143 



enzyme. The enzyme is highly specific. Of a 
large number of structurally related compounds 
tested, ethanolamine is the only compound that 
will serve as a substrate. 

The purified deaminase is pink to orange in 
color due to the presence of 1.35-2.1 equiva- 
lents of tightly bound a-adenyl cobamide de- 
rivative. The cobamide cannot be removed from 
the enzyme by charcoal or by dialysis, but it 
is removed by treatment with acid ammonium 
sulfate. In the presence of added cobamide co- 
enzyme the resolved enzyme has a higher spe- 
cific catalytic activity than the unresolved en- 
zyme suggesting that the cobamide bound to 
the enzyme is a partially degraded, catalytic- 
ally inactive compound that occupies some of 
the coenzyme binding sites. Preincubation of 
the enzyme with 10 ~ 8 M cyano-cobalamin re- 
sults in significant noncompetitive inhibition. 

The number and nature of the cobamide 
binding sites, the nature of the enzyme-co- 
enzyme interaction, and the mechanism of the 
substrate-coenzyme interaction are subjects of 
future study. 

The Metabolism of Heterocyclic Compounds 

The hydroxylation of nicotinic acid. — 
The conversion of nicotinic acid to 6-hydroxy- 
nicotinic acid is the first step in the anaerobic 
decomposition of nicotinic acid by a clostridial 
species. Purification of the enzyme that cata- 
lyzes this step is being carried out with the 
ultimate objective of obtaining a model sys- 
tem to investigate the mechanism of anaerobic 
hydroxylation of aromatic compounds. This is 
a problem of unique biochemical interest since 
the hydroxylation of aromatic compounds in 
other systems so far investigated is an obli- 
gately aerobic process involving the incorpora- 
tion of molecular oxygen into the hydroxyl 
group. 

Previous reports have shown that TPN is a 
specific electron acceptor in the anaerobic hy- 
droxylation of nicotinic acid as catalyzed by 
partially purified enzyme preparations, and 
that the enzyme catalyzed reaction is stimu- 
lated by Fe + + and by glutathione. It has now 
been found that the Fe ++ and glutathione re- 



quirements can be replaced by dithiothreitol 
plus vitamin B v > derivative. These supplements 
are apparently needed only to remove trace 
amounts of oxygen since they can all be elim- 
inated if the reaction mixture is made 
strictly anaerobic by exhaustive gassing with 
helium. In contrast to aerobic hydroxylatioD 
reactions, anaerobic hydroxylation of nicotinic 
acid is a freely reversible process. When sup- 
plemented with an appropriate TPNH gener- 
ating system the clostridial enzyme catalyzes 
the reduction of 6-hydroxynicotinic acid t< 
nicotinic acid. 

The reduction of 6-hydroxynicotinic acid 
to 6-oxo-l, 4, 5, 6-tetrahydronicotinic acid. — 
The second step in the fermentation of nico- 
tinic acid is the reduction of the double bond 
between carbons 4 and 5. The enzyme catalyz- 
ing this reaction has been partially purified 
and some of its properties determined. Re- 
duced ferredoxin appears to be the immediate 
election donor. The previously reported re- 
quirement or pyruvate and CoA in the me- 
tabolism of 6-hydroxynicotinic acid is prob- 
ably associated with the fact that in crude 
extracts of the Clostridium, pyruvate serves 
as an electron donor for the reduction of fer- 
redoxin. This conclusion is supported by the 
fact that neither CoA nor pyruvate are needed 
for the reduction of 6-hydroxynicotinic acid 
when other sources of reduced ferredoxin are 
supplied, as for example, dithionite or molecu- 
lar hydrogen plus the C. kluyveri hydrogenase 
system. Finally, it has been shown that re- 
duced ferredoxin can be replaced by low po- 
tential dyes such as reduced methyl viologen, 
but not by the reduced forms of dyes with 
greater oxidation-reduction potentials. 

The chemical synthesis of 6-hydroxynico- 
tinic acid. — Elaboration of the mechanism 
of nicotinic acid dissimilation would be greatly 
facilitated by the use of isotopically labeled 
6-hydroxynicotinic acid. Therefore, various 
methods for the chemical synthesis of this 
compound have been investigated in an effort 
to determine a practical procedure for the 
synthesis of C 14 -labeled derivatives. After sev- 
eral attempts, one reasonable method was 
developed. This method involves reaction of the 



144 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ester of nicotinic acid with benzyl bromide to 
form the quarternary salt which is then oxi- 
dized by alkaline ferricyanide to the corre- 
sponding a-pyridone carboxylic acid. By treat- 
ment with POCl 5 the a-pyridone was con- 
verted to 6-chloronicotinic acid which was 
finally hydrolyzed by strong acid to yield 6- 
hydroxynicotinic acid. 

Section on Cellular Physiology 

The research program of the Cellular Physi- 
ology Section continues to be directed toward 
the structural basis of the biochemical activi- 
ties of proteins, their biosynthesis and func- 
tional relationships in the integrated activities 
of cell structure. This program is broadly di- 
vided into three main projects: 

Studies on protein structure: In this area re- 
search is primarily focused on the fibrous pro- 
teins involved in muscle contraction and blood 
clotting with major emphasis on substructure 
and the relationship of primary structure to 
biochemical activity. 

Protein biosynthesis: Investigations in this 
area are concerned with the role of cellular 
membranes in protein biosynthesis and the 
energetics of the process. 

Biochemistry and cytology of cell transport: 
Principal attention in this area is focused on 
the cell membrane and its role in phagocytosis 
and pinocytosis. 
Summaries of the individual projects follow. 

Muscle Proteins: Myosin 

Previous reports have summarized investi- 
gations which led to development of a three 
stranded rope model of the myosin molecule 
with a globular portion at one end. The three 
strands of this model are physically and chem- 
ically identical polypeptide chains all having 
the same sense in this rod shaped molecule 
with their amino terminal ends in the globular 
portion of the molecule. This portion also con- 
tains the enzymic sites. The latter are controlled 
by two cysteine residues in each polypeptide 
chain. Methods were developed in the past for 
individually labeling these two cysteine resi- 
dues with a radioactive sulfhydryl reagent. 



Peptide fragments resulting from tryptic and 
chymotryptic digestion of the labeled protein 
have been isolated and the sequences of amino 
acids in the peptides determined. These two 
cysteine residues appear to be associated in the 
following twelve amino acid sequence: 

Cys (Si) .Gly.Asn.Val.Leu.Glu.Gly.Ile.Arg.Ile. 
Cys(Si).Arg. 

While it is not clear in this sequence how 
Cys(S 2 ) affects the enzyme activity it seems 
possible that Cys (SO influences the binding 
of the polyanion substrate by the two posi- 
tively charged arginine residues closely asso- 
ciated with Si. 

Studies on the Extraction of Actomyosin from 
Rabbit Muscle 

Actomyosin, a complex of the two proteins — 
actinand myosin — appears to provide a model 
of the functional relationship of these two pro- 
teins in the contractile process. 

While actomyosin can be prepared from its 
components, it is also extractable from muscle, 
under some conditions, particularly in pro- 
longed extraction of the tissue. The present 
study was directed at the factors influencing 
the extraction of actomyosin from muscle and 
the nature of the low molecular weight pro- 
teins associated with the preparation. In con- 
firmation of earlier proposals, it was observed 
that extraction of actomyosin occurs subse- 
quent to breakdown of the intrinsic ATP of 
muscle. Extraction of actomyosin is strongly 
inhibited by phosphate ions. In the presence 
of 0.2 M phosphate no actomyosin appears in 
the extracts even after 24 hours extraction. 
This is not due to any suppression of ATP 
breakdown, which occurs at the same rate as 
in the absence of phosphate. It appears that 
high concentrations of phosphate ions may pro- 
duce the same effect as ATP in maintaining 
the structure of the myofilaments and thereby 
inhibiting actomyosin extraction. 

In the normal extraction of actomyosin low 
molecular weight components were detected in 
association with myosin before the extracted 
protein developed the characteristics of ac- 
tomyosin. These low molecular weight com- 



NATIONAL HEART INSTITUTE 



145 



ponents appeared to be primarily either G- 
actin or F-actin of a very low degree of poly- 
merization. Their presence in a myosin prepara- 
tion, though not detectable by the usual cri- 
teria of purity, suggests the need for develop- 
ing new means of evaluating homogenety in 
this protein. 

Radiation Damage in Proteins 

The previous report described an original 
technique for trapping and labeling free radi- 
cals formed in gamma-irradiation of protein 
using tritiated hydrogen sulfide. The amino 
acids residues susceptible to free radical for- 
mation can then be identified through the ra- 
dioactive label. This technique has been applied 
to a number of proteins — ribonuclease, lyso- 
zyme, chymotrypsinogen, insulin, myoglobin, 
and gelatin. Methionine and proline were the 
residues most heavily labeled in all proteins — 
alanine, isoleucine, leucine, phenylalanine and 
valine were the least labeled. From examina- 
tion of proteolytic digests of some of the pro- 
teins, it appears that the most heavily labeled 
residues are widely distributed in the amino 
acid sequence. Experiments with unfolded — 
denatured — proteins as compared to the native 
proteins indicate that conformation plays a 
role in free radical distribution. Repair of ra- 
diation damage by reaction of the tritiated 
hydrogen sulfide with free radical also occurs 
as judged by recovery of enzyme activity and 
other properties of the native protein. 

Protein Biosynthesis 

Previous reports have demonstrated the high 
metabolic activity of the lipid soluble amino 
acid and peptide fractions of hen oviduct. In 
the current work it was found that when water 
dispersions of radioactive lipo amino acids were 
incubated in an homogenate the amino acid 
moieties were efficiently incorporated into 
homogenate proteins. This incorporation was 
more than 10 times as efficient as incorporation 
of the free amino acid and its incorporation 
was not reduced by the presence of a large 
free amino acid pool indicating a direct trans- 
fer from the lipo amino acid moiety into homo- 
genate protein. The active lipoamino acid ma- 



terial can be purified by thin layer chroma- 
tography (T.L.C.); however, a single radioac- 
tive amino acid will produce at least 9 differ- 
ent components on T.L.C., though only one or 
two of these appear to be active for incorpora- 
tion into protein. 

Nonphosphorylated Intermediates of Energy 
Transfer and Protein Biosynthesis 

Recent work in a number of laboratories 
has indicated that the electron transport sys- 
tem gives rise to high energy intermediates 
that are available to a number of mitrochon- 
dial endergonic processes, including amino acid 
uptake, prior to their involvement of inor- 
ganic phosphate leading to ATP formation. 
In the present work, the generality of this 
non-phosphorylated intermediate concept has 
been examined in anaerobically grown yeast 
in which it was found that classical uhcouplers 
of oxidative phosphorylation, dinitrophenol 
and sodium azide inhibit protein synthesis 
without inhibiting glycolysis or disturbing 
intra-cellular ATP levels. The uncouplers also 
inhibited RNA formation under anaerobic con- 
ditions, although RNA precursor pools and the 
enzyme believed to be responsible for RK syn- 
thesis, DNA dependent RNA polymerase, were 
unaffected by those agents. Although not con- 
clusive, the studies suggest an effective link of 
the synthetic activities for RNA and protein 
in the cell with transient non-phosphorylated 
intermediates of energy transfer in mitochon- 
dria or other intracellular membrane systems. 

Biochemistry and Cytology of Cell Transport 

Continuing earlier studies on the uptake of 
fatty acids by amoebae, it has been found that 
unesterified fatty acids are taken up by en- 
ergy-independent processes to give two forms 
of bound fatty acids. One is removable by 
washing the cells with serum albumin and the 
other is not. The latter class is converted to 
glycerides and phospholipids in a subsequent 
energy dependent step. 

Extending earlier studies on the chemistry 
of some of the straining and fixation tech- 
niques used in electron microscopy, the struc- 
ture of the reaction product of oxmium tetrox- 



146 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ide with oleic acid or methyl oleate has been 
determined as: 

CH 3 (CH 2 )7CH - CH(CH 2 ) 7 COOR 

V 

Qs 

CH 3 (CH 2 ) 7 CH - CH(CH 2 ) 7 COOR 

Demonstration that the osmic acid reacts 
with the double bonds in unsaturated fatty 
acids or their derivatives rather than with the 
polar portions of the molecules as widely as- 
sume, has many implications for the interpre- 
tation of electron micrographs. 

Phagocytosis by amoebae has been studied 
using polystyrene beads. Conditions were es- 
tablished in which it was possible to study 
some of the kinetics of the process, the appar- 
ent kinetic parameters being independent of 
the size of the beads, large beads being taken 
up individually and the smaller beads accumu- 
lating on the surface and being taken up in 
clusters. Phagocytosis appears to be a highly 
discriminative process, for C 14 -glucose and 
I 131 albumin present in the medium are not 
taken up with the beads. The over-all process 
is very efficient; 10'' cells (0.004 ml) can re- 
move essentially all the beads from one ml 
of medium in 15 min and at saturation the 
average amoeba has taken up about 40 beads 
of 2.6 jx diameter. The amount of membrane 
surrounding the internal beads is about equal 
to the mass of membrane surrounding the cell. 
During phagocytosis no change in cell volume 
occurs. These appear to be the first quantita- 
tive studies on the process of phagocytosis. 

Section on Comparative Biochemistry 

The activities of the Section on Comparative 



Biochemistry are centered upon the investiga- 
tion of mechanisms of lipid biosynthesis as well 
as a study of the control mechanisms in lipid 
biosynthesis. Previous work in this laboratory 
has established that the acyl carrier protein 
(ACP) plays an important function in fatty 
acid biosynthesis. Acyl groups involved in fatty 
acid synthesis are bound to ACP through thio- 
ester linkage with the sulfhydryl of the pros- 
thetic group, 4'-phosphopantetheine. The mech- 
anism of ACP involvement in this biosynthetic 
pathway has been further probed. Several of 
the reactions of this pathway have been stud- 
ied in purified systems in order to understand 
the enzymatic mechanism involved. The total 
structure of ACP, which appears to be ex- 
tremely important in determining the reactiv- 
ity of acyl-ACP derivatives with the enzymes 
of fatty acid synthesis, has been further stud- 
ied. The general occurrence of ACP in fatty 
acid biosynthesizing systems has been estab- 
lished by experiments which indicate its pres- 
ence in mammalian enzyme systems. The 
general biochemical significance of ACP has 
been further studied by a search for new reac- 
tions in which ACP may be involved. Experi- 
ments suggest that it may be involved in both 
complex lipid synthesis and sterol synthesis. 
Thus ACP is the acyl carrier in a number of 
biosynthetic reactions in which CoA was pre- 
viously thought to be involved. Therefore a 
study of the absolute concentrations of CoA 
and ACP was carried out in E. coli. In addi- 
tion studies have been initiated which should 
explain the regulation of ACP concentration 
in the cell. 

ACP Involvement in Lipid Metabolism 

Fatty Acid Biosynthesis. — ACP func- 
tions as the acyl carrier in the biosynthesis of 
long-chain fatty acids. The condensation reac- 
tion of this pathway is complex and requires 
three enzymes in E. coli: 



Malonyl-S-CoA + ACP-SH— Malonyl-S-ACP + CoA-SH 

Acetyl-S-CoA + ACP-SH— Acetyl-S-ACP + CoA-SH 

Acetyl-S-ACP + malonyl-S-ACP?±Acetoacetyl-S-ACP + CO, + ACP-SH 



(1) 
(2) 
(3) 



Sum: Malonyl-S-CoA + Acetyl-S-CoA + ACP-SH— Acetoacetyl-S-ACP + CO, + 2CoA-SH 



(4) 



NATIONAL HEART INSTITUTE 



147 



Reactions 1, 2 and 3 are catalyzed by malonyl 
transacylase, acetyl transacylase and (3-keto- 
acyl-ACP synthetase respectively. Malonyl 
transacylase was isolated as a homogeneous 
protein as shown by the presence of a single 
component upon disc-gel electrophoresis. Be- 
cause this enzyme catalyzes the transfer of a 
malonyl group from the sulfhydryl of CoA to 
the sulfhydryl group of ACP and because the 
enzyme is a sulfhydryl protein studies were 
carried out to determine whether the trans- 
acylase contains a prosthetic group such as 
phosphopantetheine. The amino acid composi- 
tion of the protein indicates that it does not 
contain this prosthetic group since there is no 
/?-ananine or mercaptoethylamine in this pro- 
tein. Thus, although the enzyme catalyzes the 
acyl transfer reaction, the sulfhydryl group of 
the enzyme involved in the transacylation is 
not a component of phosphopantetheine. 

/?-Ketoacyl-ACP synthetase was purified ap- 
proximately 400-fold, 2-fold over the previously 
reported value. This enzyme catalyzes the con- 
densation of acetyl-ACP with malonyl-ACP 
to form acetoacetyl-ACP, C0 2 and ACP-SH. 
This enzyme, like the acetyl and the malonyl 
transacylases, is a sulfhydryl protein. Since it 
had been shown previously that acetyl-ACP 
protects this enzyme against inactivation by 
N-ethylmaleimide, the hypothesis had been 
formulated that an acetyl-enzyme intermediate 
might occur during this over-all reaction. The 
400-fold purified enzyme, although still con- 
taining 4 protein components upon disc-gel 
electrophoresis, was reacted with radioactive 
acetyl-ACP under various conditions. However 
all attempts to isolate an acetyl-enzyme or 
acetyl-ACP-enzyme intermediate failed. Al- 
though acetyl-ACP must bind to this enzyme, 
the binding may not be sufficiently stable to 
allow isolation of the substrate-enzyme com- 
plex. 

Acetyl transacylase as well as the other en- 
zymes of fatty acid synthesis in E. coli are 
being studied to determine whether other pro- 
tein-bound prosthetic groups are involved in 
this pathway. 

Complex Lipid Biosynthesis. — Membrane 
preparations of E. coli catalyze the synthesis 
of monopalmitin from ^-glycerophosphate and 



palmityl-ACP. The monopalmitin was identi- 
fied by column and thin layer chromatography. 
Since glycerol cannot substitute for ^glycero- 
phosphate in this system, it is postulated that 
the primary reaction product is lysophospha- 
tidic acid which undergoes secondary dephos- 
phorylation. This same membrane preparation 
catalyzes the synthesis of lysophosphatidic acid 
from a-glycerophosphate and palmityl-CoA. It 
is not yet known whether separate enzyme 
systems are involved in the esterification of a- 
glycerophosphate by palmityl-ACP and by pal- 
mityl-CoA. 

Sterol Biosynthesis. — A possible role for 
ACP in sterol synthesis through its involve- 
ment in /3-hydroxy-/?-methylglutarate (HMG) 
synthesis is suggested in experiments recently 
carried out with yeast condensing enzyme. In- 
cubation of yeast condensing enzyme with 
acetyl-CoA and acetoacetyl-CoA gave rise as 
expected to HMG-CoA, whereas a similar in- 
cubation substituting acetoacety-ACP for ace- 
toacetyl-CoA yielded HMG-ACP. Although the 
reaction rate was faster with acetoacetyl-CoA, 
the fact that acetoacetyl-ACP was metabolized 
is significant. E. coli ACP was utilized in these 
experiments and it is known that yeast en- 
zymes discriminate against bacterial ACP de- 
rivatives. The possibility that ACP is involved 
in this reaction awaits experiments in which 
the enzyme and the ACP utilized are from the 
same source. 

Mammalian ACP 

ACP has been identified in several bacteria 
in this laboratory and in several plants by P. 
Stumpf (University of California). Its identi- 
fication in more complex fatty acid synthesiz- 
ing systems such as those of yeast, pigeons, and 
mammals is difficult since the ACP is contained 
within a protein complex in those systems. In- 
jection of C 14 -pantothenate into pantothenate 
deficient rats leads to the formation of C 14 - 
ACP within the rat fatty acid synthetase com- 
plex. This labeled ACP can thus be studied and 
perhaps isolated from the complex. Using this 
technique of in vivo labeling of ACP, it was 
previously established that the rat fatty acid 
synthetase contains protein-bound C 14 phos- 



148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



phopantetheine. Attempts to dissociate intact 
labeled ACP from this purified synthetase com- 
plex by treatment with deoxycholate, high pH, 
low ionic strength, urea, and guanidine have 
failed. Thus it is possible that ACP is part of 
the matrix of the enzyme complex in mam- 
mals. Further experiments to delineate mam- 
malian ACP will be pursued. 

Structure of E. coli ACP 

Previous studies have demonstrated that the 



E. coli enzymes of fatty acid synthesis utilize 
as substrates acyl thioesters of ACP rather than 
acyl thioesters of CoA. Since both ACP and 
CoA contain 4'-phosphopantetheine as the ter- 
minal component to which the acyl group is 
bound, it is clear that some structural feature 
other than the prosthetic group accounts for 
the fact that ACP derivatives are the pre- 
ferred substrates for these enzymes. Studies 
of the primary structure of ACP have indicated 
the following partial structure: 



(14-19) 



Ser-Thr-Ile Glu Glu Arg) (lie Glu Gly-Leu-Yal-Lys) I (Glu 

Gly 

I 
Ala 

Asp 

(24) ( ~&er- 



rAspartate peptide 
Ala-Gln-His-Gly— _Lys_ 



Lys-Ala-Met-Val-Leu Leu 

-= (56-61) -Tryptic peptide II 



4'-phosphopantetheine 



As shown above the prosthetic group is attached 
to serine which is about 24 residues from the 
amino terminus of the molecule. Peptides have 
been obtained by treating ACP with dilute 
HC1, CNBr and trypsin. Treatment of ACP 
with dilute HC1 cleaves the protein at the as- 
partic acid residue which is adjacent to the se- 
rine to which phosphopantetheine is bound. 
The resulting peptide which contains the pros- 
thetic group and the C terminus of the mole- 
cule was isolated and found to be inactive as 
tested in the malonyl-CoA-C0 2 exchange reac- 
tion. Tryptic digestion of ACP gave rise to a 
large peptide containing the prosthetic group 
and comprising about one-half the ACP mole- 
cule. This peptide was inactive as tested in the 
malonyl-CoA-C0 2 exchange reaction and in 
the /?-ketoacyl-ACP synthetase reaction. How- 
ever an acetoacetyl derivative of this peptide 
was active in the /3-ketoacyl-ACP reductase re- 
action. Kinetic studies with this enzyme have 
indicated that acetoacetyl-ACP is the best sub- 
strate, but both acetoacetyl-peptide and ace- 
toacetyl-pantetheine are active in the reaction. 
It is of particular interest that the pantetheine 
derivative is more reactive than the peptide de- 
rivative. These studies indicate the importance 



of the ACP protein structure in conferring the 
reactivity with the various enzymes of fatty 
acid synthesis. The specific sites of ACP respon- 
sible for the activity of ACP derivatives with 
these enzymes will be further studied. 

Concentrations of ACP and CoA in E. coli 

Evidence is accumulating which indicates 
that ACP rather than CoA is the acyl carrier 
in a number of biosynthetic reactions. There- 
fore it is of interest to study the concentra- 
tions of these two compounds in some biologi- 
cal system. A convenient system for this is a 
pantothenate- requiring E. coli auxotrophy. 
When this organism is grown on ^-panto- 
thenate both CoA and ACP are labeled and 
therefore can be easily measured. This auxo- 
troph was grown on different concentrations 
of C 14 -pantothenate. Cells grown on limiting 
concentrations of pantothenate contained about 
0.65 m^mole ACP/mg protein and 0.04 nut- 
mole of CoA/mg protein. When the bacteria 
were grown in an excess of pantothenate, ex- 
tracts contained 3.0 m^moles CoA/mg protein 
and 0.65 m^mole ACP/mg protein. Thus the 
concentration of ACP did not vary under these 
conditions whereas the concentration of CoA 






NATIONAL HEART INSTITUTE 



149 



was directly related to the concentration of 
pantothenate in the medium. When cells grown 
in an excess of pantothenate were transferred 
to pantothenate deficient medium, the CoA 
concentration decreased dramatically as the 
cells continued to grow normally. The ACP con- 
centration did not change under these condi- 
tions. Thus it is clear that the ACP concentra- 
tion of the cell is maintained at the expense 
of cellular CoA. These experiments also sug- 
gest that CoA can be the source of phospho- 
pantetheine for ACP synthesis. 

ACP Hydrolase 

Study of the biosynthesis of ACP and inves- 
tigation of additional reactions in which ACP 
is involved may be aided by the availability of 
ACP hydrolase (ACPase). This enzyme, puri- 
fied from E. colt, catalyzes the manganese- 
dependent cleavage of 4'-phosphopantetheine 
from ACP and thereby inactivates ACP. One 
product of this reaction is 4'-phosphopante- 
theine which has been identified by column 
chromatography. The protein product, apoACP, 
has been isolated and shown to have approxi- 
mately the same molecular weight and amino 
acid composition as ACP minus the prosthetic 
group. A hydrolytic cleavage is assumed in this 
reaction, by analogy to other phosphodiester- 
ase reactions, but a /3-elimination has not been 
ruled out. ACPase does not catalyze either the 
inactivation of any other protein that has been 
tested nor the cleavage of CoA. Its extreme 
specificity is demonstrated by its failure to 
catalyze the cleavage of 4'-phosphopantetheine 
from acetyl-ACP or from large peptides of 
ACP. It is also inactive against ACP of Clo- 
stridium butyricum, and it does not cleave 
phosphopantetheine from the mammalian fatty 
acid synthetase complex. This striking speci- 
ficity suggests that ACPase may be involved 
in the rigid control of cellular holoACP con- 
centration. 

CLINICAL ENDOCRINOLOGY BRANCH 

Studies in the Clinical Endocrinology 
Branch have included investigations of: 

A. Studies of steroidogenesis by the adrenal 
cortex, with especial reference to biogenetic 



control mechanisms, the adrenogenital syn- 
drome and the relationship of the renin-angio- 
tensin system to aldosterone metabolism. 

B. Studies of calcium and phosphorus me- 
tabolism, including in vitro studies of bone 
mineral and of solutions of calcium and phos- 
phorus, in vivo studies in rachitic dogs, and 
clinical studies in patients with metabolic bone 
diseases. 

C. Studies of renal physiology, with espe- 
cial reference to dynamic measurements and 
analysis of the renal circulation, studies of the 
amino acid clearance in cystinuria with and 
without treatment, and measurements of adre- 
nal factors affecting free water clearances. 

D. Studies of neuro-endocrine relationships, 
including measurements of factors controlling 
catecholamine excretion, studies of hormonal 
and nonhormonal disorders of taste, smell and 
hearing sensitivity, and studies of steroid ac- 
cumulation in the central nervous system. 

Studies of Steroidogenesis by the Adrenal Cortex 

Steroidogenesis was measured directly in 
vivo and in vitro in dogs. The rate of secre- 
tion of steroids into the adrenal vein as influ- 
enced by sodium deprivation, angiotensin in- 
fusion and potassium loading were measured 
over a two-day study period. The animals were 
then sacrificed and steroidogenesis was meas- 
ured in vitro with and without the addition of 
precursors. The effect of stimulation at a rate- 
limiting step was observed by measuring the 
production of steroids below and above such a 
step. In this way, it was shown that sodium 
deprivation stimulated steroidogenesis at at 
least two steps, one before the production of 
corticosterone, and another apparently between 
the corticosterone and aldosterone. The stim- 
ulus to the first of these may be angiotensin, 
but the second appears to be clearly separate 
from angiotensin. A similar technique is being 
applied to the measurement of effects of potas- 
sium and of other agents acting on steroid- 
ogenesis. 

Studies on the biogenetic defects for steroids 
in the adrenogenital syndrome were extended. 
Studies support the hypothesis that patients 
with the adrenogenital syndrome and congeni- 



150 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tal adrenal hyperplasia may have one or two 
biogenetic defects in hydroxylation of proges- 
terone or of 17-hydroxyprogesterone as sub- 
strate. When a defect is present only in hy- 
droxylation of 17-hydroxyprogesterone, there 
is hypersecretion of corticosterone and of aldos- 
terone, suggesting an actual facilitation of hy- 
droxylation of progesterone. It was further 
shown that this step was not maximally active 
in the subject on average sodium intake, but 
could be strongly stimulated by sodium depri- 
vation and that it was dependent on ACTH. 

Patients with hypertension were studied for 
involvement of the renin-angiotensin system 
by measurement of circulating renin and of 
renin secretion, as influenced by sodium intake 
and by posture, and by measurement of a wide 
variety of clinical indices, the most important 
of which appear to be the aldosterone secretion 
and metabolic clearance rate and the response 
of the serum potassium to sodium loading. The 
aldosterone secretion rate reveals the presence 
of aldosteronism and the response to sodium 
loading distinguishes those patients in whom 
over-production of aldosterone is autonomous 
in that secretion is not decreased by the sodium 
loading, and produces hypokalemia. Adrenal 
hyperplasia, autonomous in these respects, was 
found to be as common a cause of primary 
aldosteronism as tumor. In patients with tu- 
mor, renin concentration in the plasma is nor- 
mal in recumbency with average sodium intake. 

The relative role of renin and of the pitui- 
tary in the control of aldosterone secretion 
was investigated in patients with hypopitui- 
tarism. Aldosterone secretion and metabolic 
clearance rate and plasma renin were measured 
in patients with hypopituitarism before and 
after sodium deprivation. Early results sug- 
gest that effect of sodium deprivation on al- 
dosterone secretion is normal in hypopituitar- 
ism (as opposed to reported results that aldos- 
terone excretion is subnormal), but that the 
role of renin in stimulating this response is 
relatively much greater than that in normal 
subjects. Patients with panhypopituitarism are 
also under study for the effect of growth hor- 
mone upon production of renin and upon the 
secretion of aldosterone. Preliminary results 



suggest that aldosterone secretion rate is stim- 
ulated by purified human growth hormone. 

The role of renin in steroidogenesis was 
further studied by measurement of steroido- 
genesis and of plasma angiotensin in normal 
subjects during infusion of angiotensin in sub- 
pressor quantities on the one hand, and during 
deprivation of sodium on the other. Results 
suggest that angiotensin stimulates production 
of aldosterone, corticosterone and of Cortisol 
and that sodium deprivation is an effective 
stimulus to the production of angiotensin. The 
routes of secretion of circulating renin were 
further explored in normal dogs and compared 
with those found after constriction of the in- 
ferior vena cava, a procedure which increases 
renin secretion. It was found that whereas 
renal lymph delivery of renin increased mark- 
edly with caval constriction, it nevertheless 
never represented more than a very small frac- 
tion of the total renin secretion via the renal 
vein. 

Studies of Calcium and Phosphorus Metabolism 

Ionic exchange with bone mineral was re- 
explored in dynamic studies in which the time 
course of entry of calcium and phosphorous ion 
into bone mineral was explored, and the effect 
thereon of various ions in solution was meas- 
ured. In this way, it was shown that uptake 
of calcium by the crystals is isoionic and can 
be characterized as a sum of four exponential 
curves; the same constants characterized loss 
of calcium from labelled crystals. The effect of 
ions upon the exchange was found to be closely 
related to the effect of these ions upon the or- 
ganization of water molecules, and thus pre- 
sumably upon exchange at the layer of hydra- 
tion. The observations for calcium could be re- 
peated with essentially identical results with 
phosphorus. These studies are continuing with 
observation of the effect of hormones and other 
agents upon exchange. Effects of calcium ions 
on stearylphosphate monolayers are being fur- 
ther explored. The observation that calcium 
ions contract the film area per mole provides 
a direct measure of calcium phosphate inter- 
action in the solution phase. This tool may thus 
be used to explore the agents which affect this 
interaction. 



NATIONAL HEART INSTITUTE 



151 



The effects of Vitamin D on parathyroid 
hormone were further explored. In rachitic 
dogs, it was found that a state of endogenous 
hyperparathyroidism exists and removal of 
the parathyroid had its usual effects, including 
that of decreasing phosphate clearance. Ad- 
ministration of parathyroid extract promptly 
returned phosphate clearance to previous val- 
ues, despite the continued absence of Vitamin 
D. Parathyroidectomized rachitic dogs were 
further treated with Vitamin D itself, and this 
was shown to have a parathyroid hormone-like 
action in increasing phosphate clearance. The 
role of the parathyroid and of Vitamin D on 
calcium clearance is being studied in a similar 
preparation. In such animals, parathyroidec- 
tomy increases calcium clearance, a finding 
consistent with the view that rickets repre- 
sents a state of hyperparathyroidism. Whereas 
parathyroid extract was shown to be clearly 
effective in the Vitamin D deficient dogs, its 
action was quantitatively greatly augmented 
by addition of small doses of Vitamin D. The 
effects of thyrocalcitonin on renal clearance 
of calcium and phosphorus were studied in 
dogs with separate renal artery cannulas. The 
infusion of thyrocalcitonin in one side had no 
renal artery cannulas. The infusion of thyro- 
calcitonin in one side had no effect on phosphate 
clearance on that side as compared to the op- 
posite side. 

Studies in patients involve the development 
of a more satisfactory model for analysis of 
calcium accretion rate in patients subjected to 
various procedures and in patients with var- 
ious metabolic bone diseases. Compartmental 
analysis for stable and isotopic calcium was 
laborated with the help of the department of 
Biomathematical Research. These methods 
show that our investigations obtain a great in- 
crease in resolution by the prolonged observa- 
tion of radioactivity in the intact arm with a 
Packard-Armac counter. Analysis of results ob- 
tained thus far suggests that calcium accre- 
tion is markedly augmented in acromegaly and 
this result is apparent by any method of anal- 
ysis. This finding probably has no relation- 
ship to the known increase in bone formation 
rate in acromegaly, because the locus of the 
bone formation is clearlv different from the 



locus of the calcium accretion and because in- 
fusion of calcium into normal subjects produces 
equally striking increases in accretion rate. 
Studies of patients with osteoporosis receiving 
calcium 47 intravenously reveal an exponential 
decay characterized by at least three rate con- 
stants as measured over a 21-day span. The 
analysis of the curve suggests that patients 
with idiopathic hypercalciuria have a larger 
calcium "pool" than patients with osteoporosis, 
but that the loss of isotope from bones is more 
rapid with hypercalciuria than osteoporosis. 
In two patients with pseudohypoparathyroid- 
ism, together with hypothyroidism, the effects 
of parathyroid extract were measured with and 
without replacement of thyroid. In this way, 
a quantitative dependence of the action of par- 
athyroid extract upon the status of thyroid 
function was shown. Preliminary results sug- 
gest that a similar dependence of the action of 
Vitamin D upon the presence or absence of 
thyroxin wall be found in these patients. 

Studies on the role of collagen metabolism 
in bone metabolism and of the possible use of 
urinary hydroxyproline as an index of bone 
metabolism continue. Human growth hormone 
was shown to increase hydroxyproline excre- 
tion in patients with hypopituitarism, as pre- 
viously reported from several centers. Effects 
of cortisone on the rate of growth induced by 
human growth hormone and on the rate of hy- 
droxyproline excretion are under study. Stud- 
ies of the effect of calcium loading in osteopo- 
rosis have been greatly expanded to include 
measurements of the rate of bone formation 
and destruction as measured by microradiog- 
raphy and the rate of bone formation as meas- 
ured by labelling with tetracycline as well as 
the effects on calcium dynamics and the effects 
on calcium and phosphorus balance. Control 
studies are completed in three patients and in 
two, the preliminary results from the calcium 
loading experiments are available, and suggest 
a clear effect on bone dynamics. The role of in- 
testinal hyperabsorption of calcium as a deter- 
minant of hypercalciuria is under study in a 
large number of patients, with direct measure- 
ment of accumulation of isotopes in the blood 
and the bone tissue, when oral isotope is given. 
The result suggests that a syndrome of hyper- 



152 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



absorption hypercalciuria may be clearly sep- 
arable from other causes of renal stone for- 
mation. 

Studies of Renal Physiology 

Studies on the hormonal control of sodium 
and water excretion were continued in experi- 
mental animals, in normal human volunteers 
and in patients with pathological retention of 
sodium. Quantitative retention of dietary so- 
dium was produced in adrenalectomized dogs 
receiving sodium-retaining steroids by the ap- 
plication of inferior vena caval cuff. The effect 
of adrenergic ganglionic blockade was then 
measured with infusion of pentolinium. The 
pentolinium produced decreases of blood pres- 
sure and less frequently marked decreases in 
filtered sodium load; it nevertheless consist- 
ently produced moderate to marked increases 
in sodium excretion. These results confirmed 
previous results indicating a role of the adren- 
ergic nervous system in the control of renal 
handling of sodium. This was further explored 
in normal subjects and in patients with abnor- 
mal retention of sodium by the use of agents 
producing adrenergic blockade. Parahydroxy- 
amphetamine was found to decrease blood 
pressure and to lower the post Valsalva over- 
shoot of arterial pressure, and the cold pressor 
response. These results indicate that parahy- 
droxyamphetamine can function as a "false 
transmitter", replacing norepinephrine at stor- 
age sites, and producing adrenergic blockade. 
It was further found that patients subjected 
to adrenergic blockade with hydroxyampheta- 
mine lost sodium more readily and showed a 
more rapid escape from the effect of sodium- 
retaining steroids than they did before the 
drug was given. The effect of beta adrenergic 
blockade with propranolol and of alpha adre- 
nergic blockade with dibenzyline was studied 
in a number of subjects with pathologic sodium 
retention and in other subjects who were re- 
ceiving sodium-retaining steroids. Both forms 
of blockade facilitated escape from sodium re- 
tention or decrease in the quantity of edema; 
beta blockade was somewhat more effective in 
this regard than alpha blockade. Whereas 
these results again suggest a function of the 



adrenergic nervous system in renal handling 
of sodium, they further suggest that the role 
of alpha and of beta receptors in this regard 
will require reevaluation. 

The mechanism of such actions was further 
explored with direct measurements of intra- 
renal hemodynamics as measured by Xenon- 
133 . Dogs were prepared with exteriorized 
bladders and individual renal arterial cannulas 
and prepared for gamma ray counting above 
the kidney. Xenon- 133 was then injected rap- 
idly intra-arterially and the rate of decay of 
renal radioactivity measured as a function of 
time. The resultant curves were analyzed by 
computer and shown to fit well to a sum of 
four exponentials. The effects of caval con- 
striction on intrarenal blood flow were studied 
by this means and by the direct measurement 
of inulin and para animo hippurate clearances. 
The results suggest an effect of caval constric- 
tion upon intrarenal distribution of blood. The 
mechanism for the redistribution and the ef- 
fects of various vasoactive drugs on renal cir 
culation are under investigation. 

Studies with cystinuria were greatly ex- 
tended. The effects of penicillamine in systinu- 
ria were extended and compared with those of 
n-acetyl-penicillamine. Clinically, n-acetyl-pen- 
icillamine appears to induce less sensitivity 
reactions than penicillamine and to have less 
action as an anti-pyridoxine agent. Direct 
measurements of cystine excretion show that 
it has quantitatively as great or almost as great 
an effect in reducing urinary free cystine. Pre- 
vious unexplained results of penicillamine in 
decreasing total urinary half-cystine were con- 
cerned for n-acetyl-penicillamine and were ex- 
plored by (1) measurement of total excretion 
of cystine after institution of therapy, and (2) 
measurement of renal clearance of cystine, be- 
fore and after treatment with the penicilla- 
mines. In this way, it was shown that the 
decrease in total cystine excretion with the 
penicillamines does not depend upon a previ- 
ous increase in removal of the cystine by the 
urinary route and that the penicillamines con- 
sistently decrease serum cystine and have little 
effect on cystine clearance. Accordingly, the 
results establish a second role for penicillamine 
in cystinuria independent of that on the renal 



NATIONAL HEART INSTITUTE 



153 



tubules and on the sulfhydryl exchange in the 
renal tubules and in the urine. This effect man- 
ifested by a clear decrease in serum cystine 
with no change or decrease in renal clearance 
of cystine is under further investigation. Stud- 
ies in cystinosis with penicillamine have been 
instituted. The extension of these studies to 
more patients has been deferred pending the 
development of a reliable method of measuring 
cystine pool. 

The effects of adrenal factors on free water 
clearance have been studied. It was found that 
whereas small doses of sodium-retaining ster- 
oids have been shown to increase free water 
clearance while producing a corresponding de- 
crease in sodium and osmolar clearance, very 
large doses may decrease free water clearance 
while decreasing osmolar and sodium clearance 
even more markedly. These results could be ex- 
plained only as an effect of sodium-retaining 
steroids on proximal tubular sodium reabsorp- 
tion. This effect was further explored by meas- 
uring the effects of aldosterone antagonists in 
subjects under the influence of sodium-retain- 
ing steroids while maximally hydrated. In such 
subjects, (both adrenalectomized dogs and pa- 
tients with Addison's disease) blockade of the 
effect of sodium-retaining steroids with aldos- 
terone antagonists produced increases of free 
water clearance, the result which confirms a 
proximal site for some of the sodium retention 
induced by the steroid. 

Studies of Neuroendocrine Relationships 

The excretion of free and bound epinephrine 
and norepinephrine was measured in normal 
subjects as a function of the time of day. The 
effect of changes of sodium balance and of so- 
dium intake on catecholamine excretion was 
also measured. The results show a clear circa- 
dian pattern for catecholamine excretion in 
the normal with lowest values during the hours 
immediately after midnight. Acute changes in 
sodium balance induced by sodium loading or 
sodium deprivation did not produce concomi- 
tant changes in urinary bound or free epine- 
phrine or norepinephrine. These results differ 
from those reported elsewhere in which less 
specific methods for analysis were utilized. It 



is confirmed that epinephrine excretion may 
be normal in patients with adrenal insufficiency 
following adrenalectomy and that excretion of 
norepinephrine and bound norepinephrine may 
be markedly increased in Addison's disease. 

The effects of steroid hormones on nervous 
system activity were further studied. Adrenal 
insufficiency was found to produce marked 
augmentation of sensitivity of hearing which 
was represented approximately equally at all 
frequencies tested. Hearing was returned to 
normal in these patients with administration 
of carbohydrate active steroids. It was not af- 
fected by sodium-retaining steroids or by 
changes in sodium balance. 

The central nervous system of normal and 
of adrenalextomized cats was analyzed for the 
presence of corticosteroids. It was found that 
in the normal animals, corticosterone and Cor- 
tisol were found in brain, spinal cord, and pi- 
tuitary in concentrations at least ten times 
those in peripheral blood. Steroids in all these 
tissues were significantly less two weeks after 
adrenalectomy, but were still significantly 
higher than the concentrations in peripheral 
blood. Results indicate concentration of steroid 
by nervous tissue. 

The function of taste and smell was explored 
in patients with a wide variety of endocrine 
and non-endocrine disorders. In patients with 
extensive surgical resection of mandibular, 
maxillary and palatal areas, residual taste was 
measured and the results serve to extend exist- 
ing knowledge of the areas for normal taste of 
the various modalities. A number of patients 
with hypogonadism were studied and found to 
have hyposmia and an inability to taste sour 
and bitter. Patients in this group were chromo- 
somally of the XO type. A new syndrome was 
found in which facial hypoplasia, and retar- 
dation of gonadal and bone age were associ- 
ated with a decrease of recognition sensitivity 
for taste, for stereognosis of the mouth and 
for smell. 

LABORATORY OF METABOLISM 

The work of the Laboratory of Metabolism 
will be summarized here by Section. Members 
of the three Sections continue to collaborate 
productively on a number of problems, but the 



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ANNUAL KEVIEW OP INTRAMURAL RESEARCH 



bulk of the work in each group stands inde- 
pendently and is so reviewed. 

Section on Metabolism 

Mobilization and Utilization of Free Fatty 
Acids 

Metabolism of Adipose Tissue Studied 
in vitro. — An in vitro effect of thyroid hor- 
mone on adipose tissue has been demonstrated 
for the first time. Incubation of epididymal fat 
pads with triiodothyronine (1.25xl0~ 5 M) 
-significantly enhanced the response of the tis- 
sue to epinephrine, as measured by release of 
glycerol and free fatty acids (FFA). As little 
as 1 hour of incubation with hormone elicited 
a significant effect. Tissues from hypothyroid 
rats required longer exposure to the hormone, 
but after 2 hours the responsiveness to epi- 
nephrine, which is reduced in tissues of hypo- 
thyroid animals, was restored to normal. The 
thyroid hormone did not appear to influence 
the basal rate of glycerol release, but enhanced 
the responsiveness to epinephrine and also to 
ACTH and TSH. The responsiveness of the 
phosphorylase system to epinephrine was also 
enhanced in tissues incubated with triiodthy- 
ronine (T 3 ). These findings suggest that the 
effect of T 3 on adipose tissue is to "sensitize" 
the system that forms cyclic-3'5'-AMP. 

Previous studies in this laboratory estab- 
lished for the first time that prostaglandins 
inhibit the lipolytic action of epinephrine, 
ACTH and several other lipid-mobilizing hor- 
mones. Studies completed during the past year 
show that direct addition of cyclic-3'5'-AMP to 
the medium at high concentration can stimu- 
late lipolysis, compatible with the proposed key 
role of this compound in leading to activation 
of adipose tissue lipase. PGEi did not counter- 
act the effect of added cyclic-3'5'-AMP. In ad- 
dition it was shown that even high concen- 
trations of PGE! do not counteract the effects 
of high concentrations of theophyllin. The lat- 
ter compound inhibits a phosphodiesterase 
that normally breaks down cyclic-AMP. These 
results are interpreted to mean that under con- 
ditions in which the rate of cyclic-AMP for- 
mation is limiting (i.e., the low concentrations 
of theophyllin) PGE X can inhibit lipolysis, but 



that under conditions in which the further 
rate of formation is not limiting (i.e., at high 
theophyllin concentrations) PGEj becomes in- 
effective since it does not counteract the effects 
of cyclic-AMP once formed. 

Work in other laboratories has shown that 
insulin inhibits the lipolytic action of epineph- 
rine, ACTH and other lipid-mobilizing hor- 
mones, and that it acts by inhibiting produc- 
tion of cyclic-AMP. Thus, the effects of PGE X 
are closely analogous to those of insulin. For 
these reasons, the effects of PGEx on glucose 
metabolism were studied. It has been shown 
that PGEi stimulates glucose uptake by adipose 
tissue and that it enhances incorporations of 
glucose-C 14 into fatty acids. PGEi-217, a 
dehydrated derivative of PGEj, does not in- 
hibit epinephrine-induced lipolysis, and it did 
not stimulate glucose uptake. In contrast to its 
effects of glucose utilization by adipose tissue, 
PGE! did not affect glucose uptake by the rat 
diaphragm. Thus, PGEi is "insulin-like" in 
only a limited way. On the other hand, these 
findings are important in that they may clar- 
ify the fundamental basis of the action of in- 
sulin which may be related in some way to 
the adenyl cyclase system. The qualitative and 
quantitative responsiveness of this system in 
different tissues may vary, but the basic en- 
zymatic processes in the membrane may be 
closely related. 

Utilization of FFA inVivo and inVitro 

FFA Metabolism in Cell Suspension op 
Ehrlich Ascites tumor. — The convenient iso- 
lated cell suspensions obtainable by in vivo cul- 
ure of Ehrlich ascites tumors in the peritoneal 
cavity of the mouse continue to be valuable 
in studies of basic aspects of FFA uptake and 
utilization. It has been shown that under ap- 
propriate conditions ascites tumor cells can 
effect net release of FFA to the medium.To 
demonsrate release it is necessary to use very 
small concentrations of FFA in the medium (i.e. 
to treat the commercial albumin preparations 
exhaustively to remove the FFA normally at- 
tached to them). Isotopic studies show that this 
release actually occurs at all times, but under 
most conditions there is a net uptake even 






NATIONAL HEART INSTITUTE 



155 



while endogenous FFA are being released into 
the medium. This is the first example of net 
release of FFA from a tissue other than adi- 
pose tissue. The results suggest that there need 
not be any unique mechanism in adipose tissue 
for release of FFA. Instead release may be so 
striking in adipose tissue only because the ac- 
tivity" of FFA builds up to sufficiently high 
levels to permit net release in the face of 
physiologic FFA: albumin ratios in medium or 
plasma. The results, if they can be extrapolated, 
also may help to explain some paradoxical find- 
ings in isotopic studies of FFA metabolism. For 
example, the apparently greater uptake of C 14 - 
almitate over that of C 12 -palmitate in per- 
fused heart preparations may simply reflect 
exchange of intracellular unlabeled FFA for 
medium labeled FFA. The results also suggest 
that the intracellular FFA pools, low in terms 
microequivalents per gram, but highly signifi- 
cant in the total body mass, may influence the 
composition of plasma FFA and the behavior 
of labeled FFA introduced into the plasma. 

The utilization of endogenous fatty acid es- 
ters was studied by first introducing labeled 
FFA into the cells and then incubating in the 
cells in the absence of labeled FFA. Under these 
conditions, the cells break down endogenous 
lipids and a net decrease in stored lipid esters 
was demonstrated. By far the largest fraction 
utilized was drawn from stored phospholipids. 
Unexpectedly it was found that the presence 
of a high concentration of FFA in the medium 
did not inhibit the breakdown of labeled lipid 
esters. This continued at about the same rate 
as in the absence of medium FFA, a large frac- 
tion of the label being released into the medium 
as FFA. However, in the presence of glucose 
plus FFA, depletion of endogenous lipid was 
prevented and there was a net increase. Frac- 
tionation of the cell lipid into sub-classes showed 
that lecithin was the major source of fatty 
acids used in the absence of exogenous sub- 
strate. Furthermore it appears that the stored 
phospholipids do not constitute a kinetically 
homogenous pool since radioactivity decreased 
more rapidly than total phospholipid concen- 
tration. 



The presence of high FFA concentrations in 
the medium did not inhibit glucose utilization 
by ascites tumor cells. In this tissue there is 
no evidence to support the Randle hypothesis 
regarding competition between these major 
substrates. 

Studies on the Consequences of Rapid 
FFA Mobilization. — Previous studies from this 
laboratory implicate high FFA plasma concen- 
trations as relevant to a number of metabolic 
events: deposition of fat in the liver, increase 
of plasma lipoprotein concentrations, increase 
in plasma ketone body concentrations, and in- 
creases in metabolic rate of certain tissues. It 
has not been possible, however, to test the 
effects of high FFA concentration in isolation; 
i.e., it has been necessary to use hormone treat- 
ment or other devices to induce high FFA con- 
centrations. 

A method has been developed by which FFA 
can be infused in large amounts without the 
hemolytic and thrombotic implications ordi- 
narily encountered. Arterial blood from a dog 
is continuously introduced into a newly de- 
signed centrifuge (developed by IBM under 
contract with the National Cancer Institute). 
The centrifuge separates cells and plasma and 
allows each to be withdrawn separately from 
the head. This separation allows introduction 
of FFA at high concentration into the plasma 
line leading from the centrifuge. This allows 
the FFA to become bound to the albumin be- 
fore plasma and cells are recombined, thus 
avoiding hemolysis. With this method it has 
been possible to give as much as 5 grams of 
sodium oleate per hour without ill effects. 
Plasma FFA concentrations were increased 2- 
3 fold. With the development of this technique, 
it should now be possible to answer directly 
a number of interesting questions concerning 
effects of FFA concentration on other aspects 
of body metabolism. 

Factors Controlling Plasma Lipoprotein Con- 
centration 

Previous work has led to the hypothesis that 
rapid uptake of FFA by the liver in some way 
stimulated lipoprotein formation and secre- 
tion. This problem has been further studied 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



using intravenous infusions of triglyceride 
emulsions in rabbits. It has been shown that 
after a 5-hour infusion of olive oil (emulsified 
in a dilute albumin solution) during which a 
total of 2 g/kg was injected, there was a 
marked increase in the plasma concentration 
of very low density lipoproteins. That this rep- 
resented a true increase in lipoprotein concen- 
tration was established by preparative ultra- 
centrifugation and analysis of fractions and 
also by paper electrophoresis. It had been pre- 
viously postulated by other investigators that 
triglyceride emulsions induced changes in 
plasma lipid concentrations by "dissolving" 
lipids from tissue stores. If this were the mech- 
anism it should not be accompanied by in- 
creases in lipoprotein-protein but rather by a 
simple increase in the lipid content of the 
emulsion in the blood stream. The present stud- 
ies suggest that this is not the mechanism. The 
mechanism involved remains to be elucidated 
and could represent either an inhibition of the 
removal of lipoproteins or a stimulation of their 
secretion into the plasma. The latter interpre- 
tation would be consonant with the earlier 
studies mentioned above, but further work is 
needed before reaching a final conclusion. 

Metabolic Studies in Refsum's Syndrome, a 
new lipid storage disease 

Last year's progress report summarized pre- 
liminary clinical studies in an unusual neuro- 
logic disease, heredopathia atactica polyneuriti- 
formis (Refsum's syndrome), in which there 
is marked accumulation of a branched-chain 
fatty acid, phytanic acid (3,7,11,15-tetrameth- 
ylhexadecanoic acid). Continuing clinical stud- 
ies and animal studies (on the metabolism of 
phytanic acid and related branched-chain com- 
pounds) have led to the following tentative 
conclusions: 

1. Patients with Refsum's disease do not syn- 
thesize phytanic acid endogenously at any sig- 
nificant rate. There is no significant synthesis of 
phytanic acid in normal rats either from ace- 
tate or mevalonate. 

2. The metabolic error in Refsum's disease 
involves a degradative pathway for the oxida- 
tion and elimination of phytanic acid. How- 



ever, it appears that the block is incomplete 
and there is some turnover of phytanic acid. 

3. Animal studies show that both dietary 
phytol and phytanic acid, absorbed primarily 
by the lymphatic route, are potential precurs- 
ors of body phytanic acid and lead to accumu- 
lation of the latter when fed at high doses. 
Both phytanic acid (3,7,11, 15-tetramethylhex- 
adec-2-enoic acid) and dihytrophytol (3,7,11,- 
15-tetramethylhexadecanol) can be converted 
to phytanic acid in the rat, showing that there 
are two potential pathways for the conversion 
of phytol to phytanic acid. 

4. Modification of the diet of two patients 
with Refsum's disease so as to reduce the intake 
of these potential precursors (phytol, in chlor- 
ophyll, and phytanic acid, in butter fat and 
other ruminant fats) has led to a delayed but 
highly significant fall in plasma phytanic acid 
concentration. After one year on the diet, con- 
centrations have decreased by more than 75%. 
In one patient there was an increase in nerve 
conduction velocity and some increase in mus- 
cle strength, but a final determination as to 
whether this dietary treatment is curative can- 
not be made at present. These studies taken 
together establish the nature of the metabolic 
error in this new lipid storage disease. Like 
phenylketonuria, the disease appears to be one 
in which the accumulation of a metabolite 
hinges on the nature of substrates presented in 
the diet. It will be important to elucidate the 
pathway of degradation and determine wheth- 
er phytanic acid itself or one of its metabolites 
induces the nervous system dysfunction. The 
results already obtained on a restricted diet 
strongly suggest that any patients showing 
abnormal accumulation of phytanic acid deserve 
a trial with such dietary restriction. 

Clinical Studies. — In the absence of iden- 
tified case material in this country, collabora- 
tion has continued with Professor Sigvald Ref- 
sum, Professor Lorentz Eldjarn and their 
coworkers in Oslo. Normal control subjects have 
been studied in the Clinical Center, and two 
patients with Refsum's disease have been stud- 
ied in Oslo. Labeled substrates prepared here 
are shipped to Oslo, and biological samples are 
collected and returned to us for analysis. 






NATIONAL HEART INSTITUTE 



157 



(a) When these studies were begun, it 
seemed highly likely that the phytanic acid 
accumulating in Refsum's disease might be en- 
dcgenously synthesized. The basic branched- 
chain skeleton could be derived by extension 
of farnesyl pyrophosphate, a normal interme- 
diate in cholesterol synthesis, by addition of 
a fourth isoprene unit. This reaction occurs nor- 
mally in plants on the pathway for carotene 
biosynthesis. As reported last year, when 
mevalonic acid-2-C 14 was injected intrave- 
nously into a patient with Refsum's disease, 
there was a normal rate of incorporation into 
plasma cholesterol, but virtually none into 
plasma phytanic acid. In order to rule out the 
possibility that biosynthesis might be going 
on, but at a very slow rate, this patient has 
been restudied using D 2 as precursor. A con- 
stant level of D 2 in body water was main- 
tained for a period of four months. Incorpora- 
tion of deuterium into cholesterol increased 
progressively, but incorporation into plasma 
phytanic acid was minimal, at the limits of 
detectability, and showed no tendency to in- 
crease with time. The small incorporation ob- 
served corresponds to replacement of at most 2 
of the 40 hydrogen atoms in phytanic acid, and 
mass spectrometric results show that this in- 
corporation is limited to the carboxyl end of 
the molecule, compatible with non-enzymatic 
exchange at positions adjacent to the carboxyl 
carbon. On biogenetic grounds, one would ex- 
pect replacement of 14 hydrogen atoms if bio- 
synthesis occurred from acetate. The high- 
resolution mass spectrometer was invaluable 
in connection with these studies. 

(b) Studies in which phytol-U-C 14 was 
given orally to six normal subjects and to two 
subjects with Refsum's disease demonstrate a 
marked reduction in the capacity of the latter 
to oxidize it. Labeled phytanic acid was demon- 
strated in the plasma of both controls and pa- 
tients during the first day. However, in the 
normal subjects, this had virtually disappeared 
by 24-48 hours, whereas labeled phytanic acid 
persisted in the plasma of the patients and 
could still be demonstrated as late as 90 days 
after administration of the dose. The tracer 
dose was well absorbed (60-80%) and there 



was no difference between controls and pa- 
tients in this regard. 

In collaboration with Dr. Herbert Kayden at 
New York University, a patient with clinical 
manifestations virtually indistinguishable 
from those of Refsum's syndrome, but show- 
ing no phytanic acid in plasma or liver, was 
similarly studied. Results in this patient were 
comparable to those in normal control subjects. 
The possibility that this patient has a definite 
metabolic error, but one that involves a closely 
related or even identical pathway, is under in- 
vestigation. 

The conversion of tracer doses of phytol- 
C 14 to C 14 2 by the two patients with Ref- 
sum's disease was only 10-20% that of nor- 
mal subjects. Since only tracer doses were 
given, these results may underestimate the se- 
verity of the block. In fact, the prolonged per- 
sistence of labeled phytanic acid in the plasma 
would suggest that degradative capacity may 
be more severly limited. For these reasons, 
studies have been started using a "loading" 
dose of 1 gram of phytol. Normal subjects con- 
vert approximately the same fraction of this 
1-gram dose to C0 2 as they do of a tracer dose. 
Studies in patients with Refsum's disease 
using the loading dose are now in progress. 

(c) Although routine analysis of human se- 
rum lipids by gas liquid chromatography does 
not reveal the presence of phytanic acid, it has 
now been possible to demonstrate that it is 
present normally, but at extremely low con- 
centrations (0.2 mg/100 ml). A number of 
closely related branched-chain compounds have 
also been demonstrated to be present in normal 
human serum at these trace levels. In view of 
the large capacity of the normal subject to 
metabolize phytol and phytanic acid, one would 
not expect significant concentrations since the 
intake must be very low. Analysis of the Clini- 
cal Center diet shows that daily intake of phy- 
tanic acid may be about 60 mg and of phytol 
probably less than 5 mg. 

Animal Studies. — As previously reported, 
phytol is readily converted to phytanic acid by 
the rat. Similar results have now been obtained 
in the mouse, rabbit and chinchilla. In vitro 
conversion of phytol to phytanic acid has been 
demonstrated in liver homogenates. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Radioactive dihydrophytol and phytenic acid 
(the 2,3-unsaturated form of phytanic acid) 
have been prepared, and it has been shown that 
both can be converted to phytanic acid. Ani- 
mals fed phytol chronically show very signifi- 
cant tissue concentrations of phytenic acid, but 
little or no dihydrophytol. After a single oral 
dose of phytol, phytenic acid could be demon- 
strated in the lymph, but no dihydrophytol 
could be found. These results suggest that the 
major pathway may involve, first, oxidation of 
phytol to phytenic acid, and then, reduction 
of the double bond, but until kinetic studies can 
be done, this conclusion remains tentative. 

Intravenously injected C 14 -phytanic acid (in 
rats) was converted to C0 2 as rapidly as intra- 
venously injected C 14 -phytol, indicating that 
phytol oxidation may involve phytanic acid as 
an obligatory intermediate. The pathway for 
degradation of phytanic acid is being investi- 
gated in rats in vivo and in vitro. Preliminary 
results suggest that the first step in break- 
down is a decarboxylation reaction yielding 
pristanic acid. Seubert has studied the path- 
way for degradation of farnesoic acid in mi- 
croorganisms. Farnesoic acid has a 15-carbon 
branched-chain skeleton analogous to that of 
phytanic acid but includes three double bonds. 
His studies show that degradation involves a 
carboxylation reaction which is biotin-depend- 
ent. Rats were maintained on a biotin-deficient 
diet for extended periods, but their capacity to 
oxidize phytanic acid remained unimpaired. 
We have shown that the same microorganism 
used by Seubert can grow on phytanic acid as 
the sole carbon source, and studies are in 
progress to compare the degradation of phy- 
tanic acid with that of farnesoic acid. 

In an attempt to induce changes in the nerv- 
ous system like those seen in Refsum's disease, 
rats were maintained on phytol diets, which 
induce accumulation of phytanic acid in the 
blood and tissues. However, high dosages ar- 
rest growth and cause a high mortality within 
the first month or two; low dosages are toler- 
ated but as associated with only relatively 
small increases in tissue phytanic acid concen- 
tration. Rats have been maintained for up to a 
year on diets containing 0.5 by weight of 



phytol. In none of the animals thus far have 
there been changes in the retina, peripheral 
nerves, or central nervous system analogous to 
those seen in the clinical disease. However, 
because it is not possible to maintain high dose 
concentrations for extended periods of time, 
these negative results cannot be taken to rule 
out a cause-and-effect relationship between ac- 
cumulation of phytanic acid and nervous sys- 
tem changes in Refsum's disease. 

Lymphatic Absorption of Lipids 

1. The absorption of phytol, phytanic acid 
and other related substrates has been studied 
in rats in which the thoracic lymph duct was 
cannulated. It was shown that phytol and phy- 
tanic acid are both readily absorbed largely 
by way of the lymph. Phytol appears in the 
lymph mostly in combined form, only a small 
fraction being present as free phytol. Most of 
the phytol was present in a fraction shown 
to be esters of phytol and long-chain fatty 
acids. It was shown that during the course 
of absorption as much as 15% of the absorbed 
phytol was converted to phytanic acid. Phy- 
tenic acid was also demonstrated in the lymph, 
but no demonstrable dihydrophytol or any 
aldehyde derivatives were present. The capacity 
for phytol absorption in the rat is large; after 
a 300-mg dose, as much as 60 mg was ab- 
sorbed in 24 hours. Under similar conditions, 
phytanic acid was absorbed somewhat more 
rapidly, and the total absorption exceeded 100 
mg. 

Since a major source of phytol in the diet 
is that which is esterified to the porphyrin 
nucleus of chlorophyll, it was of interest to 
determine to what extent phytol is available 
for absorption. C 14 -labeled pheophytin a was 
prepared from tobacco leaves grown in C 14 2 . 
This was administered to rats by stomach tube 
and radioactivity determined in collected 
lymph, urine, C0 2 and body tissues. Less 
than 5% of the administered radioactivity was 
recovered in these fractions. Of the small 
amount of radioactivity present in the lymph, 
less than half migrated in TLC with phytol 
or phytanic acid. If these results can be ex- 
trapolated to man, it would appear that die- 






NATIONAL HEART INSTITUTE 



159 



tary chlorophyll is not likely to be a major 
source of phytol, a finding of great relevance 
in designing appropriate diets for patients 
with Refsum's disease. 

2. The origin of endogenous lipid in thoracic 
duct lymph: Even on diets devoid of fat, the 
lymph continues to show significant concentra- 
tions of lipid, but the origin of this remains 
unclear. It has now been shown that the fatty 
acid composition of thoracic duct lipid is quite 
different with respect to long-chain fatty acids 
from that of the lipid in the bile. An out- 
standing difference was the presence in lymph 
of a 20-carbon mono-unsaturated fatty acid 
which was not present in biliary lipid. The 
structure of this fatty acid has been estab- 
lished as docoso-13-enoic acid. This acid was 
also present in intestinal mucosa and in the 
intestinal contents of germ-free rats. It thus 
appears that the bile is not a major source 
of endogenous lymph lipids. Further studies 
are in progress to determine the origin of 
endogenous lymph lipids. 

Section on Molecular Diseases 

Studies of Hyperlipoproteinemia 

In 1965 the Section introduced a system for 
identification of familial disorders of plasma 
lipid concentrations based on comparison of 
lipoprotein patterns with clinical and genetic 
information. Major effort was devoted during 
the past year to further development of this 
method and its application to study of more 
kindreds. The major accomplishments have 
been as follows: 

1. An analytical sequence has been deter- 
mined that begins with a minimum examina- 
tion of the plasma by paper electrophoresis in 
albuminated buffer which segregates nearly 
all "normals" from "abnormals" and groups 
the latter in five types (I-V). When neces- 
sary to achieve firmer segregation of the ab- 
normal types, cholesterol or glyceride determi- 
nations, or a simplified quantitation of alpha 
and beta lipoproteins can be added seriatim. 

2. The rational base for this analytical se- 
quence has been thoroughly tested by compari- 
sons with other pertinent methods of separat- 
ing lipoproteins including use of immunochem- 



ical methods for an absolute definition of 
lipoprotein content. The information provided 
by this approach is superior to that obtained 
by lipid determinations alone, is feasible and 
economical for adaptation by most clinical 
laboratories, epidemiologists and geneticists, 
and cannot be obtained by any other available 
single method for lipoprotein separations, with 
the possible exception of the new computer- 
adapted analytical ultracentrifuge. (A compar- 
ison is now in progress in collaboration with 
the Donner Laboratory.) 

3. Electrophoretograms have now been ob- 
tained in more than 1500 subjects; the full 
analytical sequence has been performed in over 
700 subjects. "Normal limits" of distribution 
for four key variables have been set using 450 
normals, and about 3000 inpatient days have 
been devoted to intensive metabolic study of 
subjects with ramilial disease for correlation 
of clinical findings with abnormal lipoprotein 
patterns. 

4. Through study of 120 affected individuals 
from 45 kindreds, "familial hypercholes- 
terolemia" has been decisively split into at 
least two genotypes, which as now redefined 
are: Type II, the more common form of hy- 
perbetalipoproteinemia, and Type III, a rare 
recessive syndrome with a peculiar anomaly 
in density of beta lipoprotein. Type III, or 
"broad beta disease", had never before been 
clearly segregated from among hypercholes- 
terolemic subjects. It is associated with severe 
atherosclerosis and is more responsive to 
therapy than Type II. 

5. Increasing experience with Type IV (en- 
dogenous hyperlipemia) and Type V (mixed 
hyperlipemia) strongly suggests the presence 
of more than one mutation, some of fairly 
high frequency in Americans. For the first time 
we have detected these heritable abnormalities 
in children and have laid groundwork for bet- 
ter means of segregating the defects, including 
the establishment of normal limits for "carbo- 
hydrate induction" in terms of glyceride re- 
sponses to standard dietary loads. 

6. Cooperative projects, including the analy- 
sis in Bethesda of ten samples per week from 
the "prediabetic" population (defined by max- 
imum genetic hazard for diabetes) from the 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Joslin Clinic and all subjects undergoing cor- 
onary angiography at the Peter Bent Brigham 
Hospital, have been established. Training in 
the techniques or other assistance has been 
provided to representatives of nearly 50 groups 
in America and abroad desiring to set up the 
same system. Offering, as it does, better stan- 
dardization of nomenclature and enhanced 
recognition of familial or sporadic disorders 
having a significant association with atheros- 
clerosis and diabetes, this project is being ad- 
justed to long-range goals. 

Studies of the Structure and Function of 
Lipoproteins 

The chemical definition of lipoproteins and 
the functional interrelationships are pertinent 
to the use of lipoprotein patterns to define 
clinical disorders of fat transport. The study 
of heritable deficiencies in either beta lipopro- 
tein (abetalipoprcteinemia) or alpha lipopro- 
teins (Tangier disease) has continued to be 
valuable in this regard. Six patients with abet- 
alipoproteinemia were shown conclusively to 
have less than 1/100,000 of the normal amount 
of plasma beta lipoprotein (probably none at 
all), and four were shown to be unable to mobi- 
lize endogenous glyceride as well as from 
chylomincrons. It was concluded that beta lipo- 
protein is essential for moving glyceride out of 
cells. We have previously shown that patients 
lacking normal alpha lipoprotein can transport 
glycerides, assigning another function, yet un- 
known, to these lipoproteins. 

Using immunochemical methods, it has now 
been demonstrated in seven patients with Tan- 
gier disease (from four different unrelated 
American families) that alpha lipoprotein an- 
tigenically different from the normal alpha 
lipoprotein is present. Parents of these patients 
have both normal and "Tangier" alpha lipo- 
protein, suggesting that this disease is due to 
a mutant structural gene. The homozygous ab- 
normal is thus able to elaborate only a small 
amount of aberrant lipoprotein that fails to 
prevent cholesterol ester deposition in tissues. 
The latter was shown this year to involve even 
the skin of these patients. 

Collaborative studies with Drs. Windmueller 
of NIAMD have provided strong evidence that 



the action of orotic acid in depressing blood 
lipids, an effect which he had shown previously, 
is due to specific inhibition of the elaboration 
of beta lipoprotein by the liver. 

Studies of Tissue Lipidoses 

The discovery by Drs. Brady and Kanfer 
(NINDB) of a sphingomy elm-cleaving en- 
zyme was extended to tissues from five pa- 
tients with Niemann-Pick disease. In all the 
enzyme was drastically reduced below control 
levels, strongly suggesting that this is the 
heritable defect in this disease. With the col- 
laboration of Dr. Uhlendorf in DBS, 24 cell 
lines in tissue culture from homozygous ab- 
normals and an equal number from heterogy- 
zotes are now ready for enzyme studies. It has 
been previously shown here that the chemical 
defect is perpetuated in culture. 

Protein Structure and Function 

1. Efforts to determine the chemical prop- 
erties, relationship of structure to biological 
and immunological function, and mechanism of 
action of parathyroid hormone progressed sig- 
nificantly during the year. 

The tryptic peptides of parathyroid hormone 
were prepared and isolated and their amino 
acid composition determined. Polypeptide frag- 
ments from the hormone produced by other 
methods of cleavage, permitted the alignment 
of tryptic peptides in order, thereby providing 
a working model of the covalent structure of 
the hormonal polypeptide. By a variety 
of chemical and enzymic methods, limited 
cleavage of the polypeptide and selective mod- 
ification of individual amino acid residues were 
achieved; this permitted identification of an 
active fragment of the hormone representing 
one-fourth of the total sequence containing a 
minimum structure requisite for both biologi- 
cal and immunological activity. 

The radioimmunoassay technique has been 
extended to measurements of parathyroid hor- 
mone in the plasma of cows, goats, and sheep 
after stimulation or suppression of hormone 
production by changes in the concentration of 
plasma calcium and other ions. The rates of 
disappearance were estimated by the radioim- 
munoassay technique, for endogenous hormone 



NATIONAL HEART INSTITUTE 



161 



and for administered 131 I-labeled or unlabeled 
parathyroid hormone. These studies have per- 
mitted the calculation of the daily secretion 
rate of parathyroid hormone and the rate of 
synthesis necessary to maintain constant pro- 
duction of the hormone. It has been shown that 
plasma calcium controls parathyroid hormone 
secretion within narrow limits, hormone con- 
centration changing rapidly and markedly 
(changes of 10-15 fold) in response to physi- 
ologically or pharmacologically induced 
changes in plasma calcium. It was determined 
that parathyroid hormone secretion varies in- 
versely with serum calcium. From the calcu- 
lated regression line and other observations it 
is clear that hormone is secreted continuously 
at a basal level, with further increments in 
secretion caused by progressive degrees of hy- 
pocalcemia. It was shown that plasma phos- 
phate had no direct effect on parathyroid hor- 
mone secretion. 

Efforts have continued to develop the radio- 
immunoassay technique for routine measure- 
ment of parathyroid hormone concentration in 
human plasma. Although the technique is not 
yet satisfactory for this purpose a number of 
clinical applications have been possible. It has 
been shown that the parathyroid hormone is 
produced by nonparathyroid tumors, explain- 
ing the syndrome of "ectopic hyperpara- 
thyroidism" in patients with certain types of 
nonparathyroid malignancy. Further, the de- 
velopment of antibody causing resistance to the 
biological actions of the parathyroid hormone 
has been demonstrated in patients treated for 
prolonged periods of time with commercial 
parathyroid extract. As a possible aid in de- 
velopment of the assay for work in human sub- 
jects, human parathyroid hormone has been 
extensively purified after extraction from 
parathyroid adenoma tissue. It has been found 
that human parathyroid hormone is closely 
similar to bovine hormone in its chemical and 
biological properties. With highly purified 
human preparations available, the immunolog- 
ical reactivity of human and beef hormone can 
be compared. Use of the human hormone might 
improve the assay for work in human subjects 
through proper selection of antisera or use of 
human PTH as tracer. 



Studies have continued with evaluation of 
the effects of parathyroid hormone on ion 
transport in mitochondria. Although initially 
these studies suggested that the effect of para- 
thyroid hormone on mitochondria was highly 
selective and perhaps reflected the true mode 
of action of the hormone, more recent studies 
suggest that these effects may not be specific. 
In view of the great potential interest in the 
effects of parathyroid hormone on ion trans- 
location, studies are continuing to correlate 
the biological activity of derivatives of para- 
thyroid hormone in vivo with effects on the 
in vitro system. This should conclusively es- 
tablish the significance of the in vitro assay 
systems. 

2. Studies of the conformation and immu- 
nological properties of ribonuclease have been 
continued in further efforts to understand the 
role of protein conformation in enzymic and 
antigenic activity. Derivatives of ribonuclease 
with minor changes in covalent structure have 
been shown to have widely divergent confor- 
mational stability detectable by marked 
changes in thermally induced denaturation and 
antigenic reactivity. 

3. Collaborative studies of the treatment of 
cystinuria by use of penicillamine have been 
extended. Recent studies have further defined 
the mechanisms of action of the drug. Lack of 
change in clearance of cystine during treat- 
ment with the penicillamines and the appear- 
ance of the mixed disulfide in plasma indicate 
that the principle site of action is in plasma 
or gut, rather than in the kidney. The agent 
N-acetyl-penicillamine has been shown to be 
an even more promising therapeutic agent. 
The N-acetyl form of the drug appears to have 
a reduced potential for toxic reactions in long 
term use. 

Section on Chemistry 

The Section on Chemistiy is mainly con- 
cerned with structural analysis, synthesis and 
biosynthesis of compounds of biological origin. 
The Section also undertakes the development 
of new analytical techniques for compounds of 
biological interest. At the present, much of 
the activity of the group is centered around 



162 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



the mass spectrometer which appears to be 
generating a great deal of interest among bio- 
chemical investigators outside the Section. 
After some early difficulties, it can now be 
considered a reliable instrument around which 
biochemical experiments (e.g. isotope labeling) 
may be designed. Also, its utility in structural 
analysis is enormous (see below). In July the 
addition of a high-speed scanning system and 
tape recorder will add greatly to its value as a 
tool for structural analysis. 

Alone, or in collaboration with other groups, 
members of the Section have this year: 

1. Continued synthesis of compounds re- 
lated to phytol for studies of Refsum's 
disease, (phytanic acid, phytenic acid, 
pristane and pristanic acid). 

2. Identified pristane and phytane in rat 
liver. 

3. Identified the metabolites of 2 fluoro- 
benzoic acid. 

4. Studied the enzymatic conversion of 
GTP to 2'-deoxy-GTP and located the 
label incorporated. 

5. Identified an unusual case of hydrogen 
bonding in a 10-membered ring and 
studied the conformation of the mole- 
cule (dihydrotazettine methine al- 
cohol). 

6. Demonstrated the structure of the fla- 
vone milletine B from an Ethiopian 
fish poison. 

7. Related the structure of a new alkaloid 
astrocasidine to astrocasine. 

8. Proved the structure and absolute 
stereochemistry of astrophylline and 
synthesized a related compound. 

9. Resolved plasma kininogen (kallidi- 
dinogen) into two immunologically 
identical forms, I and II, differentiated 
by chromatographic and chemical be- 
havior. Structural work on these com- 
pounds is in progress. 

10. Developed an antiserum to kininogen I 
to assist in its purification and to an- 
swer the question whether kininogens 
I and II are present as such in human 
serum. 



11. Developed a new method of long-range 
peak matching for mass measurement 
in the mass spectrometer. 

12. Developed a solvent shift method for 
counting methoxyl groups using nmr 
spectroscopy. 

13. Investigated the hydrogen bonding of 
ortho substituted benzoic acids. 

14. Decided an important structural ques- 
tion regarding ureasterone. 

15. Solved the structure and synthesized a 
homologue of the uropygiols. 

16. Run the highest molecular weight com- 
pound ever analyzed by mass spec- 
trometry, C 72 H 24 8 F 28 N 4 P 4 (3628). 

17. Developed a new blocking group for 
ketones. 

18. Elucidated the structure of the "Sal- 
monella Resistance Factor". 

19. Completed a mass spectrometric survey 
of estrogen trimethylsilyl ethers. 

20. Elucidated the biosynthesis of a series 
of methylenebisphloroglucinols and 
found an in vitro enzyme system capa- 
ble of bringing about the critical step. 

LABORATORY OF BIOCHEMICAL GENETICS 

Nucleotide Sequences of RNA Codons 

During the past year nucleotide sequences 
of 17 RNA codons were determined and were 
assigned to amino acids or special functions. 
The sequences of virtually all codons have now 
been determined in this laboratory. The fol- 
lowing generalizations can be made concern- 
ing the nature of the code. (A) Amino acids, 
which are structurally or metabolically related 
correspond to structurally related RNA codons. 
(B) The code is logically degenerate. Recogni- 
tion of the 3'-terminal base in a trinucleotide 
is most variable and fit several synonym pat- 
terns. Alternate 3'-terminal bases of synonyms 
are as follows: U = C; G = A; U; G; U = C = A; 
and A = G (U). Degeneracy patterns were also 
found at the 5'-terminal position of trinucleo- 
tides. For example, U = C in the case of leu- 
cine and tryptophan, and U = C = A=(G) in 
the case of N-f ormyl-methionine sRNA. 



NATIONAL HEART INSTITUTE 



163 



Mechanism of Codon Recognition 

The patterns of synonym codons apparently 
define the characteristics of general codon 
recognition mechanisms. Purified sRNA frac- 
tions were used to gain further insight into 
mechanisms of codon recognition. Yeast Ala- 
sRNA of known base sequence and estimated 
to be greater than 95% pure was obtained from 
Dr. Robert Holley and was found to recog- 
nize the following synonym Ala-codons, GCU, 
GCC, GCA, and possibly also GCG. These re- 
sults demonstrate that one molecule of sRNA 
can recognize 3, possibly 4, synonym codons, 
and suggests that recognition occurs by anti- 
parallel, alternate base pairing between codons 
in mRNA and an IGC anticodon sequence in 
Ala-sRNA. 

Cells often contain multiple species sRNA 
for the same amino acid. Multiple species of 
E. coli, Val-, Ala-, and Met-sRNA were sepa- 
rated by counter-current distribution tech- 
niques and the response of each fraction to 
synonym codons was determined. The major 
peak of Val-sRNA recognized GUA, GUG, and 
GUU. This peak may represent a composite 
of two sRNA species, one responding to GUA, 
GUG and GUU, the other only to GUG. How- 
ever, a minor Val-sRNA species recognized 
only GUC and GUU. Ala-sRNAi responded 
preferentially to GCA and GCG; whereas 
Ala-sRNA 2 responded to CGU, GCC, GCA, and 
GCG. 

Met-sRNAj accepted formyl groups and re- 
sponded to UUG, CUG, AUG, and less well 
to GUG. Met-sRNA 2 did not accept formyl 
groups and responded principally to AUG. N- 
formyl-Met-sRNA apparently serves as an 
initiator of protein synthesis; selecting the 
first word to be read and phasing subsequent 
reading 

All of the available evidence suggests that 
the third (occasionally the first) base of a 
triplet can hydrogen bond with alternate bases 
in sRNA. The degeneracy patterns noted above 
result from alternate base pairing. 

Characteristics of Synonym RNA Codons 

Relative template activities and specificities 
of synonym codons were investigated by as- 



saying the formation of AA-sRNA-ribosome- 
codon complexes in reactions containing dif- 
ferent concentrations of Mg ++ and also in the 
presence of putrescine and spermidine. Syn- 
onym codons were found to differ markedly in 
both template activity and specificity. Also 
shifts in Mg ++ concentrations had a greater 
effect upon the template activity of some tri- 
nucleotides than others corresponding to the 
same amino acids. The biological consequences 
of these corresponding to the same amino 
acids. The biological consequences of these 
findings remain to be assessed. Some synonym 
codons may play special roles in protein syn- 
thesis, such as specifying the beginning or 
end of the message; others may be necessary 
for the synthesis of certain proteins or may 
selectively influence the rate of protein syn- 
thesis. 

Codon Recognition on 30 S Ribosomes 

During protein synthesis on 70 S ribosomes 
AA-sRNA may attach to both 30 S and 50 S 
ribosomal subunits. However, codon recogni- 
tion can occur on 30 S subunits only. Two 
binding sites for sRNA were found on 30 S 
subunits. AA-sRNA binding to one riobosmal 
site was dependent on K + , binding to the other 
ribosomal site did not require K + . The charac- 
teristics of each site were determined and com- 
pared to sRNA binding sites on 70 S ribosomes 

Attachment of mRNA to Ribosomes 

3 H-oligonucleotide templates were synthe- 
sized and the interaction between such tem- 
plates and ribosomes were studied. The bind- 
ing of ^-oligonucleotides of chain length 3 to 
9 was dependent upon sRNA. The use of la- 
beled oligonucleotides provides a highly sensi- 
tive technique for detecting codon recognition 
by deacylated sRNA or by a special function 
sRNA. 

Template Activty of Modified RNA Codons 

RNA and DNA contain three classes of 
codons differing in structure; 5'-terminal, 3'- 
terminal and internal codons. We previously 



164 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



showed that modification of terminal codons 
markedly affect template activity. Since such 
mechanisms may also regulate the rate of pro- 
tein synthesis in vivo, we have continued to 
explore the relation between codon modifica- 
tion and template activity. 

Trinucleoside diphosphate analogs were pre- 
pared to assess the effects of such modifications 
upon codon recognition. Substituting 5' = , 2' = , 
3'— terminal or 2'— internal ribose hydroxyls 
of oligonucleotides markedly affected their tem- 
plate activity in directing the binding of AA- 
sRNA to ribosomes. The relative template ac- 
tivity of oligo U preparations was as follows: 
p = 5'= UpUpU > UpUpU > CH 3 = p = 5'- 
UpUpU > UpUpU-3'-p > UpUpU-3'-p-OCH 3 
> UpUpU = 2', 3' = cyclic phosphate. Trimers 
with (2'-5') phosphodiester linkages, (2'-5')- 
UpUpU and also (2' = 5')-ApApA did not serve 
as templates for phenylalanine- or lysine-sRNA, 
respectively. The relative template efficiency of 
oligo A preparations was as follows: p-5'- 
ApApA > ApApA >ApApA-3'-p > ApApA- 
2'-p. 

The hexamers, UpUpUpUpUpU and ApAp- 
ApApApA were considerably more active as 
templates than the corresponding pentamers. 
These data indicate that two adjacent triplets 
are recognized by two AA-sRNA molecules 
bound to nearby ribosomal sites. 

A doublet with 5'-terminal phosphate, pUpC 
served as a template for serine-sRNA whereas 
a doublet without terminal phosphate, UpC, 
did not. Al-through the template efficiency of 
pUpC was lower than that of the triplet. 
UpCpU the data show that serine-sRNA can 
recognize pUpC. 

Universality of the Code 

Base sequences of codons recognized by 
AA-sRNA from amphibian and mammalian 
liver (Xenopus laevis and guinea pig liver, 
respectively) were almost identical to those 
recognized by corresponding E. coli AA-sRNA 
preparations. Thus, synonym codon groups are 
largely universal. However, the following 
species-dependent differences in relative ac- 
tivity of synonym codons were detected: 



eRNA 



Codon 


Bacterial 
(E. coli) 


Amphibian 

(X. laevia 

liver ) 


Mammalian 

(Guinea Pig 

liver) 


ARG 


AGG 
CGG 


+ 


+ + + + 
+ + + + 


+ + + 
+ + + + 


ALA 


GCC 
GCG 


+ 

+ + + + 


+ + + 


+ + + 

+ + 


ILE 


AUA 


— 


+ + 


+ + 


LYS 


AAG 


+ 


+ + + + 


+ + + + 


SER 


UCG 
AGU 
AGC 


+ + + + 

+ 

+ 


+ 

+ + + + 
+ + + + 


+ + + 
+ + + + 
+ + + + 


THR 


ACG 


+ + + + 


+ + 


+ + 



No differences found for additional synonyms 
corresponding to above amino acids and for 
all codons for: ASP, CYS, GLU, HIS, PHE, 
PRO, TYR, and VAL. It is possible that some 
species-dependent differences in codon recog- 
nition may selectively influence the rate of 
translation of messenger RNA. 

Regulatory Mechanisms Dependent on Viral 
Infection 

Infection of E. coli by T-2 bacteriophage 
results, within one minute, in the synthesis 
of a protein which apparently modifies one 
Leu-sRNA species present in the E. coli host. 
Simultaneously, host protein synthesis is in- 
hibited. In collaboration with N. and K. Sue- 
oka, who reported these phenomena, Leu- 
sRNA was prepared from phage infected and 
control E. coli cells and the response of each 
preparation to codons was determined. The 
modified Leu-sRNA produced after phage in- 
fection attached to ribosomes in response to 
poly UG but not to any triplet containing U or 
G, or to any other Leu-codon. The following 
hypothesis was advanced: The modified Leu- 
codon. The following hypothesis was ad- 
vanced: The modified Leu-sRNA fraction in- 
hibits E. coli, but not T-2 phage protien syn- 
thesis either by preventing the initiation of 
protein synthesis or by blocking two adjacent 
sRNA binding sites on ribosomes, and hence 
preventing further attachment of AA-sRNA to 
ribosomes. 

Studies are in progress to define possible 
consequences of selective modification of com- 



NATIONAL HEART INSTITUTE 



165 



ponents required for codon recognition. Par- 
ticular attention is being focused upon mech- 
anisms which may selectively control the rate 
of protein synthesis during viral infection and 
embryonic differentiation. 

LABORATORY OF CHEMICAL 
PHARMACOLOGY 

The Adrenergic Neurochemical Transducer 

Application of Steady State Kinetics 

It has not been generally appreciated that 
monoamines appear to diffuse from nerve end- 
ings continuously at a rate proportional to the 
amine concentration. This is not readily appar- 
ent since the amine level is maintained con- 
stant by continuous synthesis. Rates of syn- 
thesis and efflux are equal, hence 

K (rate of synthesis) = k Co 
where C is the amine concentration and k is 
the rate constant of edux. 

Proof that efflux of NE is proportional to 
NE concentration of the normal steady state 
is obtained from the decline in level after 
blockade of synthesis with a-methyltyrosine. 
In this case, 

d [NE] 
dt 
= -k[NE] and [NE] = [NE], exp (-kt) (1) 

Since synthesis is continuous (and zero or- 
der) then 

d [NE] 



dt 



K - k[NE] 



and the general expression for NE level be- 
comes 



[NE] = X" ~(x - [NE]„)exp (-kt) 



(2) 



After blockade of storage, the amine level does 
not decline to zero but to a new steady state 
concentration. The latter can be defined by the 
equation 

[NE] = 1~ 

provided k is assigned a value larger than that 
for normal organs. 

The application of kinetics to the drug- 
induced release of monoamines provides a val- 
uable tool in disclosing new facets of the behav- 
ior of nerve endings and the nature of drug 
action. In this approach, it is assumed that a 
change in amine level can result only from 
change in one of the parameters in equation 
(3), i.e., the rate either of synthesis or efflux 
is increased. With a releasing drug, the action 
is best considered as an increased rate constant 
of efflux. Furthermore, the pharmacologic ef- 
fects of a number of drugs bear a closer rela- 
tionship to the increase in rate constant of 
amine efflux than to the final steady state level. 

Turnover Rates and Times of Catecholamines 
and 5 HT 

^ At the steady state, K = k C (equation 3). 
To calculate the rate of synthesis, the rate con- 
stant k must be determined. For NE, this may 
be determined: (1) From decline in label after 
tracer doses of H 3 -NE; (2) From decline in 
[NE] after blockade of synthesis by a-methyl- 
tyrosine; (3) Rate of refilling NE stores after 
depletion by tyramine; (4) From decline in 
H 3 -NE after tracer doses of H 3 -DOPA. The 
methods all give similar results. 

Synthesis of brain 5HT is not readily blocked 
nor can 5HT be readily labeled. However, at 
the steady state 



Rate of synthesis 



(K) 

— > 



5HT 



ki 



5HIAA 



loss of 5HIAA 



hence 

K = ki [5HT] = k 2 [5HIAA]o . 
k 2 , the rate constant of 5HIAA efflux id de- 
termined from the decline of its concentration 
after blockade of MAO and from the rise after 
blockade (with probenecid) of the process that 
transports it from brain. These methods, which 
yield results similar to those calculated from 



rise in [5HT] after blockade of monoamine oxi- 
dase, have the advantage that turnover rates 
can be determined after 5HT stores are de- 
pleted by reserpine, i.e., 

K = k r [5HIAA] r 

where k r is rate constant of 5HIAA efflux, and 

[5HIAA] r is the level of acid after reserpine 

In a simplified procedure for measuring 5HT 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



turnover, each animal is used as its own con- 
trol by taking advantage of relationship 



[5HIAAlo 



ki 



[5HT] k 2 

Provided k 2 is unchanged, an increase in this 
ratio (normally about 1) signifies an increased 
5HT turnover rate. 

Results of Studies of Turnover Rates 

The turnover time of brain 5HT is about 1 
hr, showing that all the amine in granules is 
rapidly replaced without use of drugs. In con- 
trast, the turnover time of brain NE is about 
8 hr. The turnover time of NE (as well as 5HT) 
is similar in various brain regions despite dif- 
ferences in steady state levels of these amines 
in different parts of the brain. This implies 
that regional differences in concentration arise 
mainly from differences in the concentration 
of neurons that are essentially alike. The turn- 
over times of NE in peripheral tissues are also 
similar despite differing levels. 

In studies of the origin of NE nerve ending 
granules, the turnover time of NE in cervical 
sympathetic ganglion was found to be 6 times 
that in corresponding nerve endings. This 
might mean that granules in the nerve body 
are deficient in a binding component, perhaps 
ATP. 

The levels of biogenic amines are controlled 
in part by product inhibition. Thus after re- 
serpine, the formation of brain 5HT in rats 
is increased by about 30% as shown by in- 
creased steady state levels of 5HIAA. After 
blockade of MAO, when 5HT and NE levels 
rise to a plateau, rates of synthesis are almost 
zero. In view of these observations, it may be 
necessary to apply corrections to the values for 
K in the kinetic equations above. 

A problem arises in explaining why NE in 
brain of rat, rabbit and cat have about the 
same turnover time; yet after blockade of 
MAO, NE in the rat rises rapidly, in the rab- 
bit slowly, and in the cat not at all. 

Kinetics of Storage 

Releasable and resistant pools. Our observa- 
tions suggest that the common view that NE 
is stored in tyramine-releasable and tyramine- 



resistant pools is not valid. When the tyramine 
level is maintained about 3 [xg/g, NE stores 
in heart are depleted exponentially to levels 
too low to measure (T i/2-51 min) as though 
the amine were confined to a single compart- 
ment. Since the dissociation of NE from its 
complex in granules is not a rate limiting step 
in NE release (see later), the tyramine pre- 
sumably acts on the presynaptic membrane. 

Our results with tyramine are inconsistent 
with our previous studies with H 3 -NE from 
which it was concluded that the transfer of NE 
between granules and cytoplasm is the rate lim- 
iting step. From this it would be inferred that 
tyramine would deplete NE stores by 50% only 
after several hr. Improved techniques of H 3 
counting made it possible to assay tissue H 3 - 
NE, after the injection of 100 ng/kg. The level 
of 1-H 3 -NE in heart now declines as a single 
exponental (T V2-13 hr), over a period from 2 
min to 40 hr. On increasing the dose of H 3 -NE 
10-fold, a diphasic curve is once more obtained. 
In support of these results, H 3 -NE formed in 
heart and brain after injection of H 3 -DOPA 
declines as a single exponential during 1 to 40 
hr. Thus, H 3 -NE given in truly tracer amounts 
causes rapid and uniform labeling of endogen- 
ous NE. It may be concluded that NE rapidly 
equilibrates between granules and cytoplasm 
and that the loss of NE from the free pool is 
rapidly replaced by dissociation of amine from 
NE complex. 

The diphasic decline, after more than tracer 
amounts of H 3 -NE, is associated with an in- 
creased uptake of the label. The rapid half-life, 
about 2 hr, of this "excess" NE suggests that 
it gains access to some part of cytoplasm from 
which its disappearance is relatively rapid. 

The Recapture Mechanism 

Sympathetic stimulation (10/sec) of the colon 
from cats treated with phenoxybenzamine 
causes a 6- to 8-fold increase in NE over- 
flow in the venous effluent. Similar results with- 
out phenoxybenzamine are achieved by stimu- : 
lating at 30/sec. These results imply that the ; 
NE released on depolarization of the nerve ter- 
minal normally forms a complex with receptor 
sites, which acts as a brake to diffusion into 
the general circulation. When the integrity of 






NATIONAL HEART INSTITUTE 



167 



the axonal membrane is restored by repolari- 
zation, NE on receptors is recaptured through 
action of the pump. If receptor sites are occu- 
pied by phenoxybenzamine, they no longer hin- 
der the diffusion of NE, which escapes into the 
circulation during depolarization, and is no 
longer available on subsequent repolarization. 
Thus, the blocking agent augments overflow 
simply because it interferes with shuttling of 
NE between receptor and nerve ending. 

The venous overflow of NE after nerve stim- 
ulation in presence of phenoxybenzamine pre- 
sumably represents the amount of transmitter 
actually released and bound to receptors, and 
normally re-incorporated into storage sites. 
Our results show that each nerve impulse re- 
leases about 70 pg of NE/g of colon. At this 
rate, amine stores would be exhausted within 
30 min, this suggests that the important func- 
tion of the re-uptake process is to insure that 
NE stores are not depleted. 

Mechanism of NE Release by Nerve Stimu- 
lation 

The distinctive action of phenoxybenzamine 
on the re-uptake process makes it a valuable 
tool in studies of the mechanism that liberates 
NE from nerve endings. The overflow of NE 
declines on repetitive sympathetic simulation 
(10/sec), and ceases within 15 min, though 
amine stores are reduced by less than 10%. 
Moreover, most of the release occurs within 
the first 3 min. A rest period of 1 hr is ade- 
quate to restore the response to nerve stimu- 
lation. 

Since depletion of NE stores by drugs (ty- 
ramine and reserpine) can be rapid and com- 
plete, depletion by nerve stimuli must be lim- 
ited by some step not involved in drug release. 
This raises the possibility that release of NE 
by nerve stimulation is limited to vesicles in 
the vicinity of the synaptic cleft. It is possible 
that nerve stimulation, which causes influx 
of free Ca ++ , causes granules to fuse with the 
neuronal membrane. By this view, nerve exci- 
tation at a relatively high frequency would re- 
strict vesicles to vicinity of membrane. In the 
presence of phenoxybenzamine, these vesicles 
would be depleted through constant overflow. 



When stimulation is stopped, the empty vesi- 
cles at the membrane could gradually be re- 
placed by Brownian movement of fully loaded 
ones. 

Electrolyte Requirements for NE Storage and 
Uptake 

Our studies show that Na + is an absolute 
requirement for NE storage. Thus the efflux 
of H 3 -NE, when heart slices from rats treated 
with H 3 -NE are incubated with Krebs' Ringer, 
similar to that of heart in vivo (T V2-13 hr). 
Storage is markedly decreased in Na + free 
media (isotonic sucrose, Li + or choline) as 
shown by the rapid efflux of H 3 -NE. The out- 
flow is markedly decreased when sucrose is 
replaced by various amounts of Na + , approach- 
ing an asymptote in media containing 40 mM 
of Na + , as though some Na + dependent system 
were reaching a maximum rate. These results 
indicate that Na + is an absolute requirement 
in storage of NE. 

K + inhibits the effects of Na + . Thus efflux 
of NE is rapid in isotonic K + and is reduced 
by replacing K + by Na + . However, much great- 
er amounts of Na + are required to reduce the 
efflux in the presence of K", than of sucrose; 
Thus about 100 mM of Na + in the presence of 
45 mM of K + are needed before the rate of 
efflux is reduced to an asymptote. K + appears 
competitively to inhibit the process stimulated 
by Na + . 

Ca ++ is also an absolute requirement for NE 
storage since the omission of this cation 
( + EDTA) results in a rapid rate of efflux. 

Preliminary studies show that heart slices 
fail to concentrate H 3 -NE in Na + -free media, 
containing isotonic K + or Li + . These results 
are of potential importance: (1) They suggest 
that the active transport of NE is possible be- 
cause the carrier mechanism has an enhanced 
affinity for NE in the presence of Na + and 
reduced affinity in the presence of K + . This 
view would explain how NE is transported 
from an outside medium high in Na + to an 
inside one, high in K + . (2) Many of the pro- 
posed effects of electrolytes on release or syn- 
thesis of neurohormones, might well be effects 
on storage. 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Kinetics of Reserpine-Induced Release of Bio- 
genic Amines 

The action of reserpine on monoamines is 
closely tied up with the problem of their stor- 
age. Current opinion proposes two specialized 
storage mechanisms; one in granules is im- 
paired by reserpine, the second a membrane 
pump, is insensitive to reserpine but inhibited 
by cocaine. As a result nerve ending models 
proposed by other workers generally include 
the following assumptions: (1) NE formation 
is normally controlled by the rate of utiliza- 
tion; (2) NE is stored in releasable and non- 
releasable compartments; (3) NE in granules 
is stored by a specialized energy-requiring 
process which is impaired by reserpine; (4) 
NE is still taken up by nerve endings and held 
for some time after depletion of stores by reser- 
pine; (5) NE is also taken up by a membrane 
pump that is blocked by cocaine, which how- 
ever does not release the amine; (6) Tyramine, 
metaraminol and other polar phenylethyl- 
amines release NE by stoichiometric displace- 
ment from granules. 

A kinetic analysis of amine release by reser- 
pine seemed essential. In studies supporting the 
view that reserpine acts directly on granules, 
the drug added to a granule suspension pre- 
vents the uptake of H 3 -NE, but does not release 
NE. To explain why the amine is not released 
from granules, the drug is postulated to act 
by blocking the uptake of dopamine into gran- 
ules. Depletion of NE is then attributed to fail- 
ure of synthesis to replenish the physiologic 
release of the amine. 

We have shown this veiw to be untenable 
by showing that the maximal rate at which 
heart NE is released by reserpine is about 40 
times greater than that of synthesis. 

In applying kinetics to reserpine-induced re- 
lease of monoamines, we have treated the mem- 
brane carrier mechanism as a barrier to free 
flow of amine. After blockade of NE synthesis, 
even without reserpine, the amine levels de- 
cline exponentially to zero; reserpine merely 
increases the rate constant of efflux and in- 
creases the speed at which the level declines 
to zero. 



When synthesis of NE is not blocked, reser- 
pine decreases the amine level, not to zero, but 
to the new steady state value defined by 
[NE] = kC 

where k describes the slope of the exponential 
by which the amine declines to the new steady 
state value. The rate constant is maximal after 
a large dose of reserpine (5 mg/kg i.v.); the 
amine stores then decline exponentially almost 
to zero. The maximum efflux rates are rapid. 
Half lives are about 16 min for heart NE and 
about 7 min for brain 5HT, NE and dopamine. 
Calculation of free amine at nerve endings 
after complete blockade of storage is 2 to 3% 
of normal for brain NE and DA and about 
12% for 5HT. The higher level of 5HT reflects 
the greater rate of synthesis of 5HT. The high 
level of 5HT represents that available to re- 
ceptors after reserpine administration. 

The fact that reserpine, given in a maxi- 
mally effective dose, depletes monoamines at a 
maximum rate provides a common frame of 
reference in comparing the effects of reserpine. 
Thus if a dose of reserpine releases heart NE 
at a rate less than that corresponding to a half 
life of 16 min, it may be concluded that stor- 
age mechanisms are incompletely inhibited. 

The extent to which reserpine blocks the 
monoamine pump may be calculated from the 
ration of the observed rate of amine efflux to 
that after a maximally effective dose of reser- 
pine. 

In testing whether or not reserpine blocks 
the membrane pump, the drug must be given 
in maximally effective doses. Few if any stud- 
ies have shown evidence of this. Our results 
demonstrate that when H 3 -NE is injected into 
rats 4 hr after a maximally effective dose of 
reserpine, little if any label is taken up and 
retained by adrenergic neurons. Kinetic stud- 
ies provide a more rigid proof that reserpine 
completely blocks the membrane pump. H 3 -NE 
is given to rats shortly after a maximally ef- 
fective dose of reserpine followed by a MAO 
inhibitor. A few minutes later, the traces of 
H 3 -NE in heart disappear with a half-life that j 
is much shorter than that of endogenous NE 
still formed in nerve endings, showing that the 
H 3 -NE was not taken up by nerve endings. 



NATIONAL HEART INSTITUTE 



169 



Relationship of Pharmacologic Effects of Res- 
erpine to Effects on 5HT Storage 

Since the effect of reserpine on amine efflux 
is a measure of the blockade of the storage 
process, the pharmacologic effects of the drug 
may be compared with the inhibition of stor- 
age. Since previous studies indicate that block- 
ade of NE storage does not account for the 
central effects of reserpine, we compared these 
effects with 5HT storage. The results show 
that the pharmacologic effects (including se- 
dation, blepharospasm, etc.) are closely related 
to the rate constant of 5 HT efflux and that 
the most profound effects are elicited by doses 
of reserpine that alicit a maximum release. 
Doses higher than this do not increase the rate 
of efflux or the intensity of drug action. Thus 
0.9 and 5 mg/kg of drug both deplete 5HT by 
about 90%. The higher dose reduces the 5HT 
level by 90% in about 20 min; the decline after 
0.9 mg/kg is much less steep and [5HT] 
levels off in 1 hr at 75% of normal. In 4 hr 
however, the loss increases to 90%. 

False Adrenergic Transmitters 

A number of phenylethylamine analogues 
including metaraminol, 1-methyl NE, and octo- 
pamine are taken up and "stored" by adrener- 
gic neurons by a saturable process and are re- 
leased by nerve stimuli and by reserpine. A 
current misconception holds that these com- 
pounds release NE by simple physical displace- 
ment. Our results show that two separate proc- 
esses are involved. Initially the drug is taken 
up and retained by neurons, where it shares 
occupancy with NE; the drug then enhances 
permeability of presynaptic terminals, possibly 
by eliciting persistent depolarization. Our re- 
sults indicate that tyramine and guanethidine 
are also false transmitters. 

Studies of the effects of these agents, both 
in vivo, and on heart slices have provided 
further insight into their action: (1) Guan- 
ethidine, tyramine, octopamine and metarami- 
nol all release NE at the same maximum rate, 
about one-third that elicited by reserpine. (2) 
Desmethylimipramine prevents the release of 
NE by these substances (in vivo experiments). 

In exploring the possibility that these drugs 



produce presynaptic depolarization, the effects 
of guanethidine and K + have been compared. 
High levels of K + , produce an efflux of NE 
almost as rapid as that produced by guanethi- 
dine. The effects of both substances are blocked 
by bretylium and by excess Ca ++ . 

Kinetics may also be applied to the release 
of NE by the false transmitters. In this case 
the false transmitter may well increase the 
rate constant of efflux by increasing the poros- 
ity of the neuronal membrane. The rate would 
then increase with dose, approaching a maxi- 
mum as the uptake process becomes saturated. 
At the new steady state 

[NE] = -f" 

In other words, these agents need not reduce 
the NE level to zero. Even after a maximally 
effective dose of bretylium or guanethidine, 
the steady state level is 3 times that after 
reserpine. 

Relationship of Pharmacologic Effects of False 
Transmitters to Rate of NE Efflux 

In previous studies we showed that the rate 
of NE efflux is directly proportional to the 
uptake of guanethidine by adrenergic neurons. 
From these results it was suggested that the 
rate of efflux and the sympatholytic effects 
were both related to the extent of presynaptic 
polarization. Preliminary results suggest that 
the rate of NE efflux is also proportional to 
uptake of metaraminol. Failure of this drug to 
lower blood pressure may be explained by the 
direct stimulatory effect of the compound on 
adrenergic receptors. 

Effects of Desmethylimipramine (DMI) on 
Adrenergic Neurons 

A number of substances, of which DMI is 
typical, exert little action of their own but are 
potent antidepressants, block the uptake of 
catecholamines, and potentiate the actions of 
NE and amphetamine. Last year we reported 
that DMI, like cocaine, has a selective action 
in changing the permeability of adrenergic 
neurons to NE. Furthermore it prevents the 
various false transmitters from releasing NE. 
In continuation of studies of the effects of DMI 



170 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



on neuronal membranes, we have shown that 
large doses of NE displace H 3 -NE taken up 
by nerve endings in rats. If animals are first 
given DMI, the NE is still taken up by nerve 
endings but fails to displace H 3 -NE. These re- 
sults suggest that DMI acts largely on granule 
membranes and interferes with the exchange 
of the amine between granules and cytoplasm 
(or neuronal membrane). 

DMI given to rats whose NE stores are la- 
beled, reduces the spontaneous efflux of H 3 - 
NE without lowering the endogenous level. 
These results suggest that DMI reduces the 
synthesis of NE. We postulate that DMI by 
reducing the efflux of NE from granules 
(which contain little or no free amine) results 
in accumulation of free NE which then blocks 
its own synthesis. 

A possible clue to the antidepressant action 
of DMI is the marked reduction in the maxi- 
mum rate of efflux after reserpine administra- 
tion. Finally DMI added in high concentration 
to heart slices in vitro causes the release of 
NE. This supports the view that DMI has 
changed the conformation of the neuronal 
membrane. 

The Serotonergic Transducers 

Role of Serotonin (5HT) 

Precise definition of the role of 5HT in brain 
still eludes us though 5HT and NE transducers 
can be described in almost the same terms. 
Few drugs have a selective action on the 5HT 
transducer. Although our results strongly sug- 
gest that the action of reserpine is mediated 
through the continuous release of unbound 
5HT, clear proof is still lacking. 

Attempts are being made to find a role for 
brain 5HT by measuring the turnover rate of 
the amine in animals subjected to various 
physiologic states in the hope that this might 
disclose whether serotonergic pathways are 
involved. When rats are exposed to a tempera- 
ture of 38° C, brain 5HT formation is in- 
creased by 75%. This suggests that 5HT neu- 
rons in brain may be involved in control of 
temperature by dissipation of heat. 

Investigators at Pfizer & Co. have shown 
that p-chlorophenylalanine inhibits the syn- 



thesis of 5HT without eliciting pharmacologic 
effects nor counteracting the effects of reser- 
pine. In our hands, large repeated doses of the 
drug (methyl ester) depletes brain 5HT of 
rats by 80% and brain NE by 30%. The de- 
cline in 5HT is definitely related to blockade 
of synthesis since the 5HIAA level declines and 
that of 5HT fails to rise after blockade of 
MAO. The animals are hyperactive but it is 
not known whether this is correlated with the 
loss of 5HT. 

Attempts were made to establish the exist- 
ence of 5HT stores in peripheral tissues from 
the uptake of H 3 -5HT in thrombocytopenic 
rats. Preliminary results suggest that there 
may be small depots of endogenous 5HT in 
thyroid, heart, and other tissues. 

Sympathetic Target Sites 

Adipose Tissue Transducer System 

The organism makes constant adjustments 
to the environment by a unique type of adapta- 
tion in which the nervous system causes the 
almost instantaneous activation of enzyme 
systems. In this regard, adipose tissue cells may 
be considered as transducer systems in which 
the input is the sympathetic transmitter and 
the output is FFA. With these cells, we are 
particularly concerned with the way in which 
a physiological signal — a catecholamine — is 
converted to a biochemical trigger, e.g., cyclic 
AMP inside the cell. 

Last year, theophylline, which protects cyclic 
AMP from inactivation, was shown to be a 
powerful tool for studies of events in fat cells 
since its maximum lipolytic effect is 3 times 
that of NE. These studies showed that the 
activation of lipase by NE is normally limited 
by a ceiling in the steady state level of cyclic 
AMP and that theophylline, by blocking phos- 
phodiesterase, raise this ceiling causing cyclic 
AMP to accumulate to a concentration that 
produces complete activation of lipase. In sup- 
port of this view, the accumulation of cyclic 
AMP caused by theophylline was found to be 
closely associated with the lipolytic response 
and the blockade of phosphodiesterase. 

Before concluding that cyclic AMP is re- 
sponsible for the mobilization of FFA, it must 



NATIONAL HEART INSTITUTE 



171 



be shown that the nucleotide itself can actually 
increase lipolysis. Although the incubation of 
fat cells with cyclic AMP elicits only a slight 
lipolytic activity, the addition of theophylline 
in amounts having negligible activity by them- 
selves, causes lipolysis equal to that obtained 
by excess amounts of theophylline. 

Our studies indicate that cyclic AMP in adi- 
pose tissue is formed continuously, at a slow 
rate, in sympathectomized (SX) and adrenalec- 
tomized (ADX) rats. This indicates: (1) that 
NE is not necessary for adenyl cyclase activ- 
ity but merely increases it, (2) that adenalec- 
tomy does not affect adenyl cyclase, but only 
the process that activates it. 

Interaction of Sympathetic and Hormonal 
Systems 

An important accomplishment has been the 
development of a precise method for the assay 
of adenyl cyclase. With this method, changes in 
the actual amount of adenyl cyclase may be 
distinguished from activation of the enzyme 
produced by NE or ACTH. The enzyme is 
measured by the rate of production of IP- 
cyclic AMP 3 from H 3 -APT. Cyclic AMP is sep- 
arated from labeled contaminants by anion 
exchange chromatography and by treatment 
with a BaS0 4 .Zn(OH) 2 gel. The formation of 
cyclic AMP was shown to be proportional to 
time of incubation and to enzyme concentra- 
tion. The nucleotide AMP was identified by 
isotope dilution of the product itself and of the 
H 3 -5'-AMP formed by hydrolysis with puri- 
fied phosphodiesterase. 

Adipose tissue from hyperthyroid rats is hy- 
perresponsive to NE and the maximal response 
to the amine is more than doubled. This is re- 
lated to the fact that the amount of adenyl 
cyclase is more than doubled. In contrast, adi- 
pose tissue from thyroidectomized rats is poor- 
ly responsive to NE; after treatment of rats 
with triiodothyronine, the response of the adi- 
pose tissue is restored. These results indicate 
that the link between the metabolic effects of 
the sympathetic and thyroid systems is 
through adenyl cyclase. Since adipose tissue 
from euthyroid, hyperthyroid and hypothyroid 
rats give the same maximal response to theo- 



phylline, it is concluded that lipase itself is not 
affected by throid hormone. 

Adipose tissue from rats treated with corti- 
sone is also hyper responsive to NE due to an 
increase in adenyl cyclase. Again the amount 
of lipase is unaffected. However, the cortisone 
does not add to the maximum effect produced 
by thyroxin. The relationship between the per- 
missive and induction action of cortisone is 
not clear from these studies. 

After fasting for 48 hr, the adenyl cyclase is 
again increased without a corresponding in- 
crease in lipase. 

Cortisone, thyroxine, cold-exposure and fast- 
ing do not enhance phosphodiesterase, in fact 
the activity of this enzyme is increased by 
cold-exposure. 

Adrenergic Blocking Agents 

Our studies with adrenergic blocking agents 
in vitro have clarified some of the confusion in 
classifying receptor sites in adipose tissue. 
Dose-response relationships indicate that DCI, 
a beta blocking agent, competitively blocks 
NE-induced lipolysis but has little activity on 
theophylline-induced activity. Phentolamine, 
an* alpha blocking agent, acts at a different 
site since it blocks the effects of NE and theo- 
phylline to the same extent. Studies of adipose 
tissue homogenates indicate that this site is 
the lipase system itself. 

Electrolyte Requirements 

It is difficult to describe the effects of NE 
on fat cells in classical terms such as depolari- 
zation, since NE elicits almost as much lipoly- 
sis when the fat cells are incubated in isotonic 
sucrose as in Krebs' Ringer solution. The lipo- 
lytic activity of NE in isotonic sucrose is en- 
hanced by the action of small amounts of K + . 
On increasing the [K + ] the lipolytic activity 
of NE is progressively diminished, and is ab- 
sent at a [K + ] of 100 to 150 mM. This inhibi- 
tory effect of K + is not counteracted by Na + . 
The absence of Ca ++ reduces NE lipolysis by 
only about 50%. 

In considering the component parts of the 
adipose tissue transducer system, drugs may 
act on adirose tissue in a number of ways: (1) 
Displacement of NE from receptor sites, 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



(DCI); (2) Interference with membrane de- 
polarization of cell by NE (adrenalectomy ?); 
(3) Inhibition of adenyl cyclase (no drug yet 
demonstrated); (4) Direct activation of adenyl 
cyclase (catecholamines); (5) Induction of 
adenyl cyclase (thyroid, cortisone); (6) Inhi- 
bition of cyclic AMP action; (7) Inhibition of 
phosphodiesterase (theophylline); (8) Activa- 
tion of phosphodiesterase (nicotinic acid); 
(9) Inhibition of lipase system (phentol- 
amine; (10) Shifts in K + — insulin. 

Studies on Chemical-Induced Shock 

Previous reports from this laboratory have 
shown that ADX rats and rats whose adren- 
ergic function is blocked fail to respond to ex- 
ternal stimuli that require an increased 
expenditure of energy. The failure of epi- 
nephrine to elicit sympathetic responses in 
ADX animals indicates that their incapacity to 
withstand cold or strenuous work results from 
failure of sympathetic target organs to respond 
to transmitted messages. Communications are 
re-established by aldosterone as well as gluco- 
corticoids, suggesting that the inexcitability of 
adrenergic receptors after adrenalectomy is 
related to changes in electrolytes. 

We are now studying substances, categori- 
cally classified by Selye as stressors, and known 
to be much more toxic in ADX than in normal 
animals. We hope that the study of shock, in- 
duced by drugs whose mechanism of action is 
known, might lead to a better understanding of 
clinical shock. We started this study with the 
limited objective of establishing whether the 
toxicity of these stressor agents is enhanced in 
SX animals as well as in ADX animals. Our 
results show histamine and endotoxin, in doses 
that are not lethal to intact rats, are almost 
100% lethal to both ADX and SX rats. 

Treatment of ADX rats with a glucocorticoid 
or with epinephrine-in-oil provides partial pro- 
tection against the lethal effects of histamine 
and endotoxin; complete protection is provided 
by giving both substances. Treatment of SX 
rats with epinephrine, alone, provides complete 
protection against the lethal effects of histamine 
and endotoxin. 

Both histamine and endotoxin produce a 
blood pressure drop presumably by dilating the 



small blood vessels. The lethal effects of these 
substances in ADX and SX animals might 
stem from their action on the microcirculation. 
In normal animals, protection against hypo- 
tension is provided by signals sent to the brain 
via the baroreceptors, and the resultant 
adrenocortical discharge. In SX animals, there 
are no catecholamines to be discharged; in ADX 
animals, the catecholamines have little effect 
on the sympathetic system in the absence of 
glucocorticoids. 

The lethality of formalin and tourniquet 
trauma is also greatly increased in ADX rats. 
Thus doses of formalin or a degree of tourni- 
quet trauma that cause no deaths in control 
animals are 100% lethal in ADX rats but the 
toxicity is not enhanced in SX rats. Moreover, 
treatment of ADX rats with a glucocorticoid 
provides complete protection against the lethal 
effects of formalin and trauma. DOCA also 
affords considerable protection. Thus the lethal 
effects of formalin and tourniquet trauma are 
not mediated by the circulation. The results are 
consistent with the view that formalin and 
tourniquet trauma produce a toxic agent which 
is responsible for the toxic effects. Glucocorti- 
coids would act by preventing the formation 
of this agent rather than by overcoming its 
effects. In support of this view, is the well- 
known fact that the lethal effects of tourniquet 
trauma occur only after removal of the tourni- 
quet. 

The Nonmast Cell Histamine Transducer 

All tissues contain considerable amounts of 
histamine not in mast cells; in fact in some 
species including rabbit, cat (except for skin) 
and man, mast cell stores comprise only a 
small fraction of total histamine in the body. 
Last year we reported results which have led 
us to the provisional conclusion that nonmast 
cell histamine mediates exocrine secretions. 

Selective Labeling of Nonmast Cell Histamine 

The view that parenterally administered 
H 3 -histamine selectively labels nonmast cell 
stores has now been fortified by studies show- 
ing that the specific activities of histamine in 
rat gastric mucosa and cat salivary gland are 



NATIONAL HEART INSTITUTE 



173 



almost indentical with those of amine released 
by cholinergic agents into saliva and gastric 
juice. From these results it may be inferred 
that the decline in radioactivity accurately re- 
flects the endogenous turnover of histamine 
and that the release of histamine can be calcu- 
lated from the release of radioactivity. 

The assay of H 3 -histamine by isctopic dilu- 
tion has shown that the second part of the 
diphasic disappearance of radioactivity is arti- 
factual and results from tritiated water pres- 
ent as an impurity in the injected H 3 -hista- 
mine as well as from that formed by exchange 
in the bcdy. Corrected values now reveal that 
turnover of H 3 -histamine in various exocrine 
glands corresponds to a half-life of about 1 hr 
and in skeletal muscle of about 4 hr. 

Distribution and Fate of Injected H '-Histamine 

In rats, the highly polar metabolite present 
in various tissues has been identified as a con- 
jugate, presumably the riboside, of histamine 
itself and not of the acid as reported by others. 
In the cat, the uptake of H 3 -histamine is also 
high in exocrine glands and occurs to some 
extent in all tissues. In exocrine organs H 3 - 
histamine is rapidly converted to methylhista- 
mine which is also retained by tissues. H 3 - 
histamine in the cat is excreted in 2 phases; 
at first the urine contains considerable amounts 
of free histamine and methylhistamine. The 
rapid decline in the excretion of these amines 
coincides with their disappearance from plas- 
ma. At this time, the urinary excretion of the 
acid metabolites remains high. Since studies 
with tissue slices show that deamination oc- 
curs largely in kidney and liver, these results 
suggest that the bases are released into blood 
largely unchanged and are then deaminated 
by kidney and liver. 

Methylhistamine injected into the submaxil- 
lary gland, intra-arterially, elicits some saliva- 
tion; placed in lumen of cat stomach it elicits 
gastric secretion. These findings raised the 
question whether methylation of endogenous 
histamine leads to its inactivation or whether 
the substance is normally stored and has a 
physiological role. 



Various cat organs contain considerable 
amounts of a substance that, like methylhista- 
mine, is chloroform extratcable, deaminated 
by diamine oxidase, and reacts with dinitro- 
fluorobenzene to form a derivative with a 
similar R f value. However, mass spectrogra- 
ph^ (Dr. Highet, Lab. of Metabolism) of the 
substance indicates that it is not methylhista- 
mine. The distribution of the "apparent" 
methylhistamine is of interest since it is high 
in brain stem and certain exocrine glands but 
absent from cerebellum and skeletal muscle. 

The specificity of the histamine uptake 
mechanism was studied by measuring the 
uptake of H-methylhistamine in rats. The pat- 
tern of uptake and disappearance of methyl- 
histamine closely resembles that of H 3 -hista- 
mine. From current evidence we favor the view 
that methylhistamine is not normally formed 
and retained, and that exogenous methylhista- 
mine may act like a "false transmitter". 

Release of H '-Histamine 

Histamine appears to have an important 
role in exocrine glands. Thus stimulation of 
parasympathetic nerves or administration of 
acetylcholine or its analogues cause the release 
of histamine into the secretions of submaxil- 
lary gland, gastric mucosa, bile and pancreas. 
Pilocarpine also causes a marked efflux of H 3 - 
histamine into these secretions. Gastrin and a 
factor in saliva cause the efflux of H 3 -hista- 
mine into gastric juice. Secretin induces the 
efflux of H 3 -histamine into bile and pancreatic 
secretions. 

Finally preliminary results indicate that 
stimulation of skeletal muscle is associated with 
the release of H 3 -histamine. 

Synthesis of Histamine 

Last year we reported evidence that the syn- 
thesis of histamine in the salivary gland was 
regulated by utilization. Additional studies 
show that after ligation of the esophagus to 
deprive the rat stomach of the stimulatory ef- 
fects of saliva, gastric secretion is reduced by 
about 90% and the turnover of histamine by 
about 75%. 



174 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Factors That Affect Drug Action 

Passage of Substances Across Membranes 

Central nervous system. Since the pharma- 
cologic effects of polar drugs are often studied 
after intraventricular injection, it is impor- 
tant to understand the nature of the boundary 
between ventricles and brain substance. After 
intraventricular injection, C 14 labeled inulin, 
sucrose and mannitol enter brain tissue at 
rates roughly proportional to their diffusion 
coefficients in agar gel of infinite thickness. 
Such studies might make it possible to esti- 
mate the fraction of the ependymal surface 
permeable to large lipid-insoluble molecules. 

Further studies have been made of the ac- 
tive transport system in rat brain which trans- 
fers 5HIAA directly from brain to blood. Last 
year it was reported that this process is com- 
pletely blocked by probenecid. The transport 
system is also blocked by dinitrophenol given 
in hyperthermic doses. In addition it is mark- 
edly impaired when rats are exposed to a tem- 
perature of 38° C for 16 hr. It is possible 
that such impairment is a sensitive indicator 
of brain damage. 

Intestinal absorption. Highly liposoluble sub- 
stances like DDT and dieldrin are absorbed in 
part via the lymphatic circulation of the in- 
testine. The substances are absorbed by the fat 
transport process of the intestinal epithelium, 
presumably due to their association with lipid 
micelles. 

Biliary excretion of drugs. Carboxylic acids, 
sulfonic acids and chlorothiazide are secreted 
into bile by the same transport mechanism. 
Probenecid, a carboxylic acid, which markedly 
inhibits the biliary secretion of organic acids, 
is itself excreted in bile and appears to act 
competitively. The choleretic activities of pro- 
benecid and chlorothiazide are indirect effects 
stemming from the large quantities of water 
they take into bile by their osmotic effects. 
Ouabain, an active inhibitor of many trans- 
port mechanisms, is itself secreted into bile 
by a process that differs from those which 
secrete organic acids and bases. In addition, 
ouabain is also taken up by liver slices by an 
active transport process. 



Thus the liver transports organic com- 
pounds by at least three general processes; 
one for acids, one for quarternary and per- 
haps tertiary amines, and one for ouabain and 
presumably other glycosides. 

Enzymatic Mechanism of Membrane Transport 

Research is directed toward a molecular- 
physiologic explanation of processes carried 
out by cell membranes. Two sorts of processes 
await explanation: (1) the active transport 
mechanisms which utilize energy to move ca- 
tions, glucose, amino acids, catecholamines 
and other substances across various cell mem- 
branes; (2) processes by which nerve stimuli 
induce transient changes in conducting mem- 
branes and neurohormones produce transient 
permeability changes at receptor sites. 

It is hoped that studies of the Na + , K + , 
ATPase from beef brain will provide a clue 
to the mechanism by which ATP energy is 
used for cation transport. Attention has been 
shifted to the active center of this enzyme. 
Previous work has identified as an acylphos- 
phate, a phosphorylated intermediate whose 
formation is catalyzed by Na + . We have at- 
tempted to determine the concentration of 
transport sites in the membrane by "trapping" 
the acylphosphate as a stable hydroxamate. 
The results suggest that Na + might cause two 
effects: (1) a reaction with ATP to yield the 
protein-bound acylphosphate and (2) a con- 
formational change that protects the acyl de- 
rivative from reacting with hydroxylamine. 

Attention is now focused mainly on the K + - 
stimulated phosphatase step as the point 
where most of the ATP energy is utilized for 
the transolocation of ions. We have sought 
artificial substrates which interact specifically 
with the K + -sensitive step. p-NPPase which 
catalyzes the K + -stimulated hydrolysis of 
p-nitrophenylphosphatase resembles ATPase in 
certain respects; after adrenalectomy the ac- 
tivities of both enzymes in kidney are de- 
creased and are restored by corticosterone. 
Differences in ouabain inhibition, however, 
show that the two enzymes are different. 
p-NPPase may provide a model for the study 
of Reactivated systems; more recent studies 
suggest that acetylphosphate may serve as a 



NATIONAL HEART INSTITUTE 



175 



substrate for the K + -sensitive step of the gen- 
uine transport ATPase. 

Studies have shown that aldosterone does 
not act by changing the synthesis of trans- 
port ATPase. Thus aldosterone exerts drug 
effects on transport in the toad bladder but 
has no effect on ATPase or on enzymes in- 
volved in ATP synthesis. It is possible that 
aldosterone induces the synthesis of a mem- 
brane component controlling the diffusibility 
of Na + . 

An attempt is underway to identify adrener- 
gic receptors by labeling with covalently fixed 
blocking agents. Experiments in connection 
with these studies have shown that reserpine 
produces a 5-fold increase in submaxillary 
glycoprotein in the denervated as well as in 
the innervated gland. The possibility that this 
action is mediated by the pituitary-adrenal 
system is under investigation. 

Enzymatic Mechanisms of Drug Metabolism 

Enzyme processes. Microsomes contain a 
cytochrome which in its reduced form com- 
bines with CO to form a complex; the cyto- 
chrome is called P-450 because the complex 
with CO has an absorption maximum at this 
wavelength. P-450 in microsomes is reduced 
by TPNH and then reacts with 2 to form 
an "active 2 " complex which transfers 2 
to various drugs. In the absence of drug sub- 
strate, this complex breaks down to H 2 2 . 
P-450 also participates in the anerobic re- 
duction of nitro and azo compounds. These 
general reactions are illustrated below 



TPNH cytochrome C 




— reductase 




TPNH > P-450 


> O2-P-450 — > Oxidation 


X 


I of Drug 


Reduction of 


H 2 2 


nitro and azo drugs 





P-450 is a component of the TPNH-depen- 
dent enzymes in microsomes that are responsi- 
ble for the hydroxylation, dealkylation, deam- 
ination and desulfuration of drugs and for at 
least one enzyme that catalyzes sulfoxidation. 

TPNH cytochrome c reductase is now im- 
plicated as a component of P-^450 reductase 



by studies showing that the reactions which 
utilize drugs and TPNH-cytochrome c re- 
dictase have practically the same Km's with 
respect to TPNH. In addition, 2'-adenosine 
phosphate inhibits the reductase and the drug 
reactions to the same degree. 

Small but reproducible changes in the ab- 
sorption spectrum of liver microsomes are pro- 
duced by substrates and inhibitors of drug- 
metabolizing enzymes, even in the absence of 
TPNH. These changes are of two types. In 
presence of substrates, the dissociation con- 
stants for these changes closely agree with 
Km's (substrate) of the oxidation reactions, 
suggesting that they represent the formation 
of enzyme-substrate complexes. Substances that 
inhibit drug oxidation appear to act either by 
displacing substrate from the enzyme or by 
interfering with the flow of electrons between 
TPHN cytochrome c reductase and P^450. 

Induction of microsomal phospholipid by 
phenobarbital. The increased oxidation and 
reduction of drugs produced by phenobarbital 
and other foreign compounds is associated with 
increased amounts of protein and phospholipid 
in liver microsomes. The increased content of 
protein can be explained by an increased rate 
of protein synthesis. However, studies of the 
incorporation of P-32 suggest that the in- 
crease in phospholipid is caused by a decrease 
in phospholipid catagolism. 

Mechanism of Teratogenesis by Thalidomide 

The mechanism of thalidomide-induced tera- 
togenesis in laboratory animals, is still un- 
known. We have now shown that about 3% 
of the thalidomide in rabbit fetuses is bound 
to tissues irreversibly, mainly with nuclear 
RNA and DNA. This incorporation may be a 
characteristic of rapidly developing tissues, 
for 2 to 3 times as much labeled thalidomide 
is incorporated into nuclear RNA and DNA 
of rapidly growing rat liver (after partial 
hepatectomy) as in the liver of normal rats. 
These findings make it likely that thalidomide 
causes teratogenicity by acylation of RNA and 
DNA or its precursors and suggest that alky- 
lating agents and thalidomide cause tera- 
togenic effects through similar mechanisms. 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Such a reaction would explain our findings 
that differences in the embryo toxicity of 
thalidomide in the rabbit and the rat are 
minimized when the drug is administered in- 
travenously since the effects are likely to be 
related to a high level of drug action over a 
short time. By giving the drug intravenously, 
reproducible effects are obtained in rabbits 
with doses ranging from 2.5 to 10 mg/kg and 
in rats with doses of about 10 mg/kg. 

Pharmacogenetics 

The problem of individual variability in re- 
sponse to pharmacologic agents is receiving 
much attention because increasingly large 
populations are being exposed to drugs. Since 
each of the multiple enzymes that control drug 
metabolism is genetically controlled, mutations 
may lead to toxicity through accumulation of 
the drug (acatalasia, atypical cholinesterase 
and slow isoniazid inactivation). 

Our studies are focused on the basic ques- 
tion whether inbred animals respond more 
uniformly to a drug than outbred animals (Fi 
generation hybrids). The total variability of 
the strain is reduced, providing that genetic 
components of variation, such as color and 
antigenic variation predominate over environ- 
mental effects. Reports on responses to bar- 
biturates are conflicting. Some claim that the 
response of inbred mice is more variable than 
that of outbred mice, others that it is less. 

Our results show that hexobarbital elicits a 
sleeping time that, in some inbred strains of 
mice, is more variable than in outbred mice, 
whereas in others it is equal and in still others 
it is less. These variations are attributable 
largely to differences in rate of drug metab- 
olism. On recovery of righting reflex, the 
brain hexobarbital level is similar in all strains, 
indicating that the CNS sensitivity to the drug 
is remarkably uniform. 

Strain differences in hexobarbital metabo- 
lism may be related to participation of multiple 
forms or the isozymes of a given enzyme. Lac- 
tate dehydrogenase (LDH) which exists as 5 
isozymes in most body cells, provides us with 
a model of how these forms result when 2 dis- 
similar subunits combine randomly to form 



active polymers. The synthesis of the sub- 
units may be controlled by gene activity, but in 
addition the catabolism of enzymes may be an 
important variable in determining distribution. 
The relative stabilities of Pure LDH 1 and 5 
were studied as possible variables in their bio- 
logical regulation. Stability to heat was differ- 
entially altered by pH, ionic strength, NADH, 
oxaloacetate, malate, and fructose-1-6 diphos- 
phate. The results show that substances may be 
fixed to allosteric sites thereby protecting the 
molecule from inactivation or accelerating heat 
denaturation. Three distinct allosteric sites 
have been identified on LDH 1 and 5. 

Clinical Studies with Desmethylimipramine 

An NIH-sponsored collaborative study with 
the Karolinska Institute is providing a par- 
tial answer to an important question: Are in- 
dividual differences in metabolism of liposolu- 
ble drugs used in treatment of mental diseases 
large enough to account for individual differ- 
ences in side effects and efficacy. In this study, 
plasma levels of the antidepressant drug des- 
methylimipramine (DMI) are determined by 
the isotope derivative technique dveloped in 
this laboratory (see methods) by Dr. W. Ham- 
mer, a participant in this project. The results 
indicate: 

1. Plasma levels of patients on the same 
dosage regimen differ by as much as a 
factor of 20. 

2. Thus far, side-effects are seen only in 
those patients who metabolize the drug 
slowly and who have the highest plasma 
level. 

3. In general therapeutic effects on depres- 
sion are associated with the concentra- 
tion of DMI in the plasma. 

4. Patients shortly after treatment with bar- 
biturates have very low levels of DMI. 

5. The side-effects in two patients were 
characterized as "extreme anxiety". No 
detectable DMI was found in the plasma 
of these subjects and subsequent studies 
have shown extreme anxiety to be a re- 

6. The psychiatrists collaborating in this re- 
search now insist on plasma levels of 
flection of lack of treatment. 



NATIONAL HEART INSTITUTE 



177 



DMI; for the first time they feel that 
they can control therapy and toxicity. 

Development of New Methods of Analysis 

The ever increasing potency of modern 
drugs, especially those used in the treatment 
of mental disease, has created the need for 
more methods of higher sensitivity for the 
assay of organic compounds. 

The isotope derivative technique, described 
in last year's report has been applied to the 
routine assay of desmethylimipramine and has 
yielded particularly rewarding results. This 
method is based on the extraction from plasma 
of desmethylimipramine and its subsequent 
acetylation with H 3 -acetic anhydride of high 
specific activity. The method has been modified 
so that as little as 5 ng/ml of drug can be 
accurately measured. 



A potentially simpler approach for the rou- 
tine assay of drugs in ng amounts is the use 
of gas liquid chromatography with an electron 
capture detector. This sensitive device responds 
to organic compounds containing halogen or 
nitro groups at levels of about 10 ~ 15 M. If a 
drug does not contain these groups they may 
be introduced by forming a derivative of the 
compound after its extraction from plasma. 

A method for assay of amphetamine, the- 
oretically applicable to other aliphatic pri- 
mary amines, involves condensation with 
Dansyl chloride (the demethylamino analogue 
of naphthalenesulfonychloride) to yield a 
highly fluorescent derivative. 

A method has been developed for the assay 
of methylhistamine in biological tissues. In 
this procedure methylhistamine is assayed as 
the dinitro-fluorobenzene derivative. 



NATIONAL INSTITUTE OF ALLERGY AND 
INFECTIOUS DISEASES 



Introduction 

The year began with Dr. Leon Jacobs con- 
tinuing as Acting Scientific Director. In late 
September Dr. John R. Seal joined NIAID as 
Associate Director for Intramural Research. 
Dr. Seal had retired from the United States 
Navy on September 1, 1965, after serving in 
various capacities in the Navy medical re- 
search program during the major part of his 
military career. Dr. Jacobs remained as a con- 
sultant to the Director, NIAID, until January 
1, 1966, when he retired from the Commis- 
sioned Corps of the U.S. Public Health Service 
and joined the Division of Biologies Standards 
NIH. These transitions had little effect on the 
momentum of the broad-based intramural re- 
search program. More serious in prospect was 
the retirement or resignation of other pro- 
gram leaders, and as the year closed the In- 
stitute was recruiting for a new Clinical Di- 
rector with the need to restaff the Laboratory 
of Clinical Investigations to a large degree, 
facing needs to restructure and redirect its 
research effort related to tropical medicine in- 
volving changes in the programs in tropical 
virology and parasitology, and seeking leader- 
ship for the medical mycology research pro- 
gram. 

Despite these unsettling influences, the year 
was a remarkably productive one with a con- 
tinuing output of scientific publications re- 
flecting the leadership role the NIAID Intra- 
mural Research Program holds in many aspects 
of infectious diseases and immunology. 

Dr. Karl Habel was selected to give the An- 
nual Dyer Lecture at the National Institutes of 
Health. Drs. Habel, Leon Jacobs, Robert Hueb- 
ner, and Robert Coatney were awarded Dis- 
tinguished Service Medals by the United 



States Public Health Service for their scien- 
tific contributions. Dr. Maurice Landy received 
the DHEW Superior Service Award. Many 
other honors were afforded the scientific staff 
as are detailed in the reports of the individual 
laboratories. 

A new parameter was added to the responsi- 
bilities of the NIAID Intramural Research 
Program with the assignment of Chairmanship 
of the NIH Cholera Advisory Committee to 
Dr. Seal. This responsibility includes the sci- 
entific management of the SEATO Cholera 
Research Program and the Pakistan-SEATO 
Cholera Research Laboratory in Dacca, East 
Pakistan. The SEATO Cholera Research Pro- 
gram was developed as a result of the spread 
of cholers in Southeast Asia, the Pacific, and 
westward from its traditional endemic focus 
beginning in 1958, and includes the sponsorship 
of scientific symposia for exchange of infor- 
mation, grants, training contracts, and re- 
search in cholera in SEATO member countries 
with basic funding by the Agency for Inter- 
national Development (AID) and scientific 
management by NIH as a result of several 
AID-DHEW, AID-NIH interagency agree- 
ments. The Pakistan-SEATO Cholera Research 
Laboratory in East Pakistan is sponsored under 
the umbrella of the Program and a government 
of Pakistan-AID agreement but receives addi- 
tional funding and support through a NIH- 
PL 480 research grant and from several 
SEATO member countries for conduct of chol- 
era research. Among its more notable contri- 
butions during this year have been informa- 
tion on the high degree of effectiveness of 
United States manufactured cholera vaccine 
in the prevention of cholera, the long duration 
of vaccine induced immunity, particularly in 



179 



180 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



adults in cholera endemic areas, added data on 
the value of tetracycline in the therapy of 
cholera along with comparative data on the 
lesser effectiveness of several other antibiotics, 
new information on non-cholera diarrheas and 
on a malabsorption syndrome which occurs in 
Americans subsisting on a local diet, and rec- 
ognition of hypoglycemia as a complication of 
cholera in infants. 

The Board of Scientific Counselors of NIAID 
was asked to undertake a review of the entire 
viral research program. Departing from the 
usual custom, three meetings were held dur- 
ing the year, one at the Middle America Re- 
search Unit (MARU), Canal Zone, Republic of 
Panama, and another meeting featuring a re- 
view of the "slow" or chronic virus disease 
research sponsored by the NINDB, the Ex- 
tramural Research Program of NIAID, and the 
Rocky Mountain Laboratory. One important 
area in which the advice of the Board had been 
sought was that of arbovirus research. The 
Board emphasized the National needs for NIAI 
intramural research to continue a strong pro- 
gram in arbovirus research and recommended 
that the excellent small program at the Mid- 
dle America Research Unit (MARU) be 
strengthened and extended both by the as- 
signment of a more broadly based mission and 
staff to MARU and by more active scientific 
collaboration with the Gorgas Memorial Lab- 
oratory and university-based research groups. 
The Board also recognized the leading role of 
the Rocky Mountain Laboratory in research on 
animal models of slowly developing, progres- 
sive viral infections causing neuropathies, pul- 
monary, vascular, and renal disease, the prob- 
able relevance of the work to certain human 
diseases, and the probable inability of aca- 
demic institutions to play any prominent role 
in this type of research during the next dec- 
ade. It recommended strengthening of the viral 
research competence of the Rocky Mountain 
Laboratory and installation of new facilities to 
permit inoculation of material of human ori- 
gin, particularly brain specimens from hu- 
mans dying of "Kuru," into the animal species 
being studied. 

The importance of the Intramural Research 
Program to the Collaborative Research Pro- 



gram of the NIAID was more evident as the 
demands on the intramural scientific staff in- 
creased. The Laboratories of Infectious Diseases 
and Tropical Virology undertook the testing of 
many of the reagents being developed in the 
Research Reference Reagents Branch, pro- 
vided seed stocks of viruses, developed new 
methodology and trained some contractor per- 
sonnel in their use, site-visited contractors' in- 
stallations, and provided informed members to 
advisory committees. The Laboratory of Infec- 
tious Diseases was especially involved in the 
Vaccine Development Branch with several of 
the staff making important contributions as 
project officers and participants in the field 
testing of new vaccines such as the oral 
adenovirus type 4 vaccine and parainfluenza 
vaccines, research for further definition of the 
oncogenic potential of various adenoviruses, 
initial testing of vaccine against mycoplasma 
pneumoniae, developments in methodology, and 
advisory services to the Vaccine Development 
Committee. The Laboratory of Immunology pro- 
vided very similar support to the Transplan- 
tation Immunology Branch. 

The variety and scope of the Intramural Re- 
search Program is illustrated in the selected 
examples of research which are summarized 
in the following pages. 

LABORATORY OF CLINICAL 
INVESTIGATIONS 

A major emphasis of the laboratory has 
been in the further study of respiratory virus 
dynamics in the infection of human volun- 
teers derived from the Federal prison system. 
Other research on the mechanisms of fever 
and host responses in various syndromes, in 
immunogenetics, in parasitology, and in lep- 
rosy has continued. 

Transmission Dynamics in Respiratory 
Infections 

Using Coxsackie A-21 virus as a model, ex- 
tensive studies were made in human volun- 
teers of transmission dynamics. Quantitative 
studies were carried out on the presence of 
virus in respiratory tract secretions, the role 
of breathing, speaking, coughing and sneezing 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



181 



in release of viral particles to room air, the 
effect of particle size in creation of viral aero- 
sols which remain in suspension in room air for 
long periods of time and on the transmission 
of infections via the airborne route to other 
volunteers. Coughing- was shown to be the most 
important source of airborne viral particles 
and human-to-human transmission via the air- 
borne route conclusively demonstrated. Air- 
borne transmission of Coxsackie A-21 virus 
infections between artificially infected volun- 
teers and other non-immune volunteers under 
dormitory conditions was also demonstrated. 
Virus was recovered from the dormitory air 
by use of large volume air sampling tech- 
niques during these trials. 

Remarkably small doses of virus were infec- 
tive when aerosolized in small particles and 
this was particularly true in adenovirus in- 
fections. The human infectious dose (HID 50 ) 
was about 30 TCID 50 for Coxsackie A-21, 1 
TCID, a for adenovirus type 4, and 0.68 TCID S „ 
for rhinovirus. Even smaller doses given by 
nasal drops caused similar illness in the case 
of Coxsackie virus and rhinovirus. A larger 
dose of adenoviris by nasal drops was neces- 
sary to infect and only the aerosol route regu- 
larly produced typical illness. 

Antibody in Nasal Secretions and Serum 

By challenge experiments in volunteers the 
apparent greater importance of 9S-14S anti- 
body in resistance to rhinovirus infection than 
7S or 19S antibody was demonstrated. 11-14S 
antibody was shown to appear in nasal secre- 
tions, tears, and saliva after its first appear- 
ance in sera and was associated with only yA 
immunoglobulin in these secretions. This evi- 
dence strengthened the hypothesis that yA 
antibody in secretions results from an active 
selective transport mechanism between serum 
and secretion but does not rule out a possible 
local production of antibody. 

Chilling and Respiratory Infection 

Controlled experiments were made on the 
effects of chilling with and without an actual 
decrease in body temperature on the course 
of rhinovirus infections. No evidence could be 



obtained that chilling before, during, or sub- 
sequent to the clinical disease in any way al- 
tered its course. 

Adenovirus Soluble Antigens 

Purified hexon and fiber antigens from ade- 
noviruses types 1 and 4 were administered to 
volunteers with good neutralizing antibody re- 
sponse in a high percentage of subjects. The 
7S antibody response was similar in degree 
and duration to that observed in human sub- 
jects given infectious virus. 

Influenza 

Influenza was produced in volunteers with 
three TCID- )0 doses of A2 virus when given as 
an aerosol. Low levels of homologous neutraliz- 
ing antibody did not protect against infection 
with this small dose nor did heterologous anti- 
body in higher titers. A/Equi 2 virus caused 
a high incidence of infection but infrequent 
illness in human volunteers. Serial passage of 
equine influenza virus in man did not increase 
its pathogenicity for man or decrease its path- 
ogenicity for equines. Their findings suggested 
that A/Equi 2 virus is rather host specific 
and that horse-to-man transfer under natural 
conditions is probably a rare event. 

Mechanisms of Fever and Host Responses 

Extensive studies are in progress relating to 
the pathogenesis of fever, reticuloendothelial 
function and immunological reactivity of hu- 
mans and experimental animals. Investigations 
are being carried out in patients with famil- 
ial Mediterranean fever, recurrent fever of un- 
known etiology, leprosy, a variety of neoplasms 
and normal volunteers. 

Through utilization of a specific and sensi- 
tive assay for etiocholanoalone, it has been 
shown that increased plasma levels of this 
steroid are not correlated with febrile episodes 
in a variety of patients. The administration 
of this pyrogenic steroid is proving useful as a 
tool for studies of bone marrow reserve, granu- 
locyte kinetics and fever. 

A bentonite flocculation test was developed 
for the demonstration of circulating anticryp- 



182 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



tococcal antibodies in patients with cryptococ- 
cosis. 

Patients with Hodgkin's disease have an en- 
hanced phagocytic capacity. Likewise, patients 
with lepromatous leprosy have an increased 
reticuloendothelial clearing capacity in addi- 
tion to a variety of immunological defects. 

It was also shown that passively transferred 
endotoxin tolerance persists for at least 3 
weeks despite rapid decay of passively trans- 
ferred antibody. 

Systemic Fungus Infection 

Attempts to improve chemotherapy of sys- 
temic mycoses met with mixed success. In vitro 
tests of Nocardia suggested two potentially 
useful antibiotics: ampicillin and capreomycin. 
A combination of amphotericin B and hy- 
droxystilbamidine appears promising in the 
treatment of mice infected with North Ameri- 
can blastomycosis. Intraventricular adminis- 
tration of amphotericin B by use of a new 
prosthesis has not been encouraging. Hamycin, 
a new antifungal antibiotic, was dropped from 
clinical trials because of poor chemotherapeu- 
tic effect. 

Factors affecting the cure of cryptococcosis 
with amphocericin have been the subject of 
continuing study. The role of immunologic 
paralysis, the occurrence of delayed hyper- 
sensitivity, and the means of adaptation by 
Cryptococcus to chemotherapy have been eval- 
uated with respect to their effect on prognosis. 

Leprosy 

The leprosy program continued with the 
evaluation of rimino compound B.663, in the 
treatment of lepromatous leprosy. The patient 
under study is of particular interest because 
he had received no prior or concurrent sul- 
fone therapy. Increasingly precise bac- 
teriologic techniques have confirmed the anti- 
biotic potency of B.663 against the leprosy 
bacillus. 

In addition, experience suggests that B.663 
possesses an anti-inflammatory action against 
erythema nodosum leprosum reactions. These 
reactions constitute the most objectionable fea- 
ture of sulfone therapy, which is currently 



standard, and B.663 may prove to have wide 
application used in combination with the sul- 
fones. 

Laboratory of Infectious Diseases 

The efforts of this laboratory continue to be 
focused in several major programs: 

1. Cancer and leukemia virus studies. 

2. The viral and mycoplasma causes of 
acute respiratory diseases. 

3. Rubella. 

4. Epidemiology of picoranaviruses and 
eosinophilia meningitis in Oceania. 

5. Bacterial metabolism and physiology. 

6. Medical mycology. 

The virus research groups work in close re- 
lationship with the National Cancer Institute 
on the problems of oncogenic viruses and with 
the National Institute of Neurological Diseases 
and Blindness on Rubella and related problems. 

Adenoviruses as Oncogenic Agents 

Eight of 31 human adenoviruses have been 
shown to cause cancers in hamsters (types 3, 
7, 12, 14, 16, 21, and 31). The first and chief 
indication that the tumors induced by adeno- 
virus inoculation were actually directed by the 
adenovirus genes was the discovery of non- 
virion complement fixing (CF) antigens in the 
virus-free tumor cells. Similar nonvirion anti- 
gens, now called T antigens or neoantigens 
occur in cells infected with adenoviruses. These 
antigens were shown to be new proteins which 
were synthesized by the input virus before 
new viral DNA appeared. The T antigens there- 
fore represent a revolutionary new category 
of virus induced antigens. Many different lines 
of research here and in other laboratories con- 
firmed these findings not only for adenoviruses 
but for other DNA viruses, such as SV40 and 
polyoma. 

Tumor and T Antigens as Possible Determinants 
of Adenovirus Oncogenesis 

The presence of virus-specific but non-virion 
T antigens in cells infected and/or trans- 
formed by human adenoviruses were confirmed 
for oncogenic strains of simian, canine and 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



183 



bovine adenoviruses. Thus, the regular pres- 
ence of T antigens in adenovirus tumors sug- 
gested that such antigens may be determinants 
of oncogenesis, a hypothesis that was supported 
by remarkable correlations between groupings 
of the adenoviruses according to oncogenic po- 
tentials, Green's G + C DNA base composi- 
tions, Rosen's hemagglutinins (HA), and T 
antigens. 

Adenovrus-SV40 Hybrids 

Exciting new discoveries on the transfer of 
SV40 and adenovirus genetic information from 
the adenovirus type 7-SV40 hybrid to other 
adenoviruses were made. Not only was the 
SV40 genome transferred from type 7 to type 
2, but the genome of type 7 responsible for 
stimulating production of the virus specific T 
antigen was transferred as well. These obser- 
vations proved without question that viral 
DNA was responsible for the synthesis of the 
non-virion T antigens. 

Adenovirus-Associated Viruses (AAV's) 

A new defective virus, dependent entirely 
on adenoviruses acting as helpers for replica- 
tion, was discovered almost simultaneously by 
workers at Baylor University, The University 
of Pittsburgh and the Laboratory of Infectious 
Diseases. The AAV virus was found to be the 
smallest of the known viruses, measuring no 
more than 15 m^ in diameter. Its DNA was 
shown to be double stranded and to have a 
base composition much different from that of 
the adenoviruses from which they were de- 
rived. 

Also, recently discovered were two addi- 
tional serologically distinct AAV's. The AAV's 
are quite unique among mammalian viruses; 
however, similar wholly dependent "satellite" 
viruses are found among the plant viruses. 

Leukemia 

Extensive studies with several animal models 
continued. It was shown that infectious virus 
was obtained when hamster tumor cells result- 
ing from Bryan Rous Sarcoma Virus (defec- 
tive virus) were grown in mixed culture with 
chick embryo fibroblasts. When such mixed cul- 



tures were injected into RIF-free chicks, non- 
infectious sarcomas resulted which had the 
karotype of chicken cells and CF antigens of 
the Rous Sarcoma cells. Tissue cultures de- 
rived from these sarcomas remained free of in- 
fectious virus until an avian leucosis virus was 
added, following which Rous Sarcoma Virus 
appeared. In other studies, a quantitative test 
for murine leukemia viruses was developed as 
was an in vitro assay system for mouse (Mo- 
loney) Sarcoma Virus. 

Vaccine Against Mycoplasma Pneumoniae 

A protective effect of a formalin inactivated 
M. pneumoniae vaccine was demonstrated in 
volunteers. Men who developed antibody fol- 
lowing inoculation of the vaccine resisted chal- 
lenge with a virulent suspension of the or- 
ganism, whereas illness occurred in 10 of 13 
in unvaccinated controls. 

Mycoplasma Epidemiology 

Considerable advances were made in under- 
standing the epidemiology of Mycoplasma 
behavior of this organism a longitudinal study 
of seven training platoons was made at the 
Parris Island, S.C., Marine Recruit Training 
Center. Fifty to 74% of recruits possessed M. 
pneumoniae growth-inhibition antibody at the 
start of training. Among the antibody nega- 
tive (i.e., susceptible) recruits the infection 
rate for the 14-week training period was quite 
high — 60 to 80%. Growth-inhibition antibody 
correlated well with resistance to infection, 
but it was not completely protective since ap- 
proximately 20% of recruits with pre-existing 
anibody became infected during training. The 
latter finding provides the first evidence that 
reinfection with M. pneumoniae occurs under 
natural conditions. 

Mycoplasma — Fundamental Studies 

Three major advances were made in under- 
standing these organisms. First, the hemolysin 
of M. pneumoniae was found to be a peroxide. 
This finding has implications in the pathogene- 
sis of mycoplasma-induced disease as well as 
in autoimmune phenomena induced by myco- 
plasma. Second, the DNA homology technique 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



revealed the genetic distinctness of the myco- 
plasmas from bacteria. This finding clearly es- 
tablished the mycoplasmas as a separate group 
of microorganisms, a group with its own iden- 
tity. Third, the protective antigen of M. pneu- 
moniae was defined as a lipoprotein. The lipid 
hapten of this complex was further defined as 
a low molecular weight phospholipid contain- 
ing glycerol, four amino acids and several fatty 
acids. The chemical structure of this protec- 
tive antigen is nearing definition. 

Host Resistance to Respiratory Disease 

In volunteers challenged with type I parain- 
fluenza virus, serum antibody did not correlate 
well with resistance to infection. In contrast, 
antibody in nasal secretions did correlate well 
with resistance to infection. Such antibody 
proved to be the decisive factor in determin- 
ing the outcome of experimental challenge with 
type I parainfluenza virus. An inactivated type 
I virus vaccine stimulated serum but not nasal 
secretion antibody. Such vaccinated individuals 
did not resist experimental challenge with the 
virus. However, antibodies developed in the 
nasal secretions of volunteers following infec- 
tion and such individuals resisted rechallenge. 

Studies in Oceania 

Picornaviruses have been found to be fre- 
quent causes of infection among adults and 
children in Honolulu. The exact nature of the 
problem remains to be defined since the Cox- 
sackie Group A related viruses recovered are 
not pathogenic for suckling mice and neutral- 
izing antibody cannot be studied. 

An outbreak of Dengue in French Polynesia 
druing 1964-65 has been further analyzed and 
indicates that this was the first introduction 
of an arthropodborne virus into the island in 
the life span of individuals studied. 

More evidence has been obtained on the re- 
lationship between A. cantonensis and eosino- 
philic meningitis in studies in Thailand and 
Oceania. The probable source through inges- 
tion of raw shellfish, fish and vegetables has 
been indicated. Recent outbreaks in French 
Polynesia apparently resulted from contami- 
nated lettuce. 



Rubella Virus 

These studies are supervised by Dr. John 
Sever of NINDB and Dr. Huebner of NIAID. 
In collaboration with Drs. A. Fabiyi, G. Git- 
nick, L. White, R. J. Hildebrandt, and D. A. 
Fuccillo, they are studying the role and be- 
havior of rubella and other viruses as causes 
of perinatal disease and defects. 

Rubella virus given to pregnant ferrets early 
in gestation produced runting and abortion 
of the newborn. Neutralizing antibodies de- 
veloped in all inoculated animals, showing that 
active infection had been produced. Newborn 
ferrets given rubella virus develop a chronic 
infection lasting 6 to 8 weeks. The newborn 
and pregnant ferret represents the first and 
as yet only animal study system available for 
rubella virus other than primates. Should the 
runting and abortion prove to be due to infec- 
tion of fetal tissues, this system may prove ex- 
tremely useful in the study of birth defects. 

Rubella virus syndrome is also being studied 
clinically, serologically and epidemiologically. 
Preliminary data derived from the NINDB 
Perinatal Study groups have provided a pro- 
file of the immune status to rubella among 
women of childbearing age. Considerable geo- 
graphical variations were observed; antibodies 
were found in 85% of the women in the east- 
ern part of the U.S., in 70% on the west 
coast and in less than 50% in Hawaii. 

A new rubella vaccine development program 
was initiated in collaboration with NIAID's 
Vaccine Development Branch with Dr. John 
Sever, NINDB, as the major project officer on 
a number of contracts. This program is only 
in the beginning stage; however, several types 
of vaccines produced on commercial contracts 
should be available for field testing early in 
FY 1967. Reference virus pools and antisera 
needed for evaluation of vaccines have been 
produced in this laboratory and distributed to 
the various contractors. 

Various methods for increasing the titer of 
rubella virus have been successful, and these 
have been utilized for producing complement 
fixation test antigens and also seed stocks for 
candidate rubella vaccines. 



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185 



Bacterial Metabolism ami Physiology 

Hydrogenomonas eutropia has been utilized 
in model system study of electron transport 
mechanisms because of its ability to utilize 
molecular hydrogen. Further evidence was ob- 
tained on the role of cytochome c but the exact 
enzyme which mediates transfer of electrons 
has not as yet been defined. Solution of this 
problem will enable studies on the role of 
organic nutrients in metabolism in auto- 
trophic and heterotrophic cells. 

Studies on the role of iron and siderophilin 
in the growth and metabolism of Staphylo- 
coccus aureus continue. Marked physiological 
differences exist between iron-deficient and 
iron-rich cells of S. aureus and these are addi- 
tionally altered by growth of such cells in the 
presence or absence of glucose. Iron-deficient 
cells vary in the following characteritsics: poor 
growth, imparled respiration, increased gly- 
colytic activity, decreased catalase activity, a 
reduction in cytochromes and cytochrome oxi- 
dase, and decreased sensitivity to cyanide in- 
hibition. Iron seems to act as an inductor of 
electron transport and respiratory enzyme for- 
mation. There have also been further studies 
on the enzyme which releases teichoic acid 
from S. aureus cell walls. This is produced in 
varying amounts by nine different strains and 
has been purified 10-fold in one preparation. 
Na + , Mg + + and cobalt are required for activity. 
If further purification can be achieved and the 
linkage point of attack identified, removal of 
teichoic acid from the cell wall may permit 
identification of the role of this acid in cell- 
wall replication and in the binding of certain 
antiobiotics. 

Studies on the structure, replication and ge- 
netics of streptococci also continued. These in- 
clude characteristics of L-form colonies. By 
electron microscopy, small intravescular mas- 
ses of elementary bodies in the order of 0.05/j. 
are seen but filtration studies have failed to 
demonstrate colony forming capacities of units 
smaller than 0.22^. in size. The surface recep- 
tor site of Group C streptococci has been identi- 
fied but the phage receptor site in Group A 



streptococci remains unidentified despite inten- 
sive efforts to identify its location and nature. 
The coexistence of two M antigens in the cell 
wall of Group A streptococcus has been con- 
firmed and the occurrence of a type 12 M pro- 
tein in a Group C streptococcus observed. Ef- 
forts to achieve laboratory transduction of 
Group A streptococci have not as yet been suc- 
cessful but continue since this apparently oc- 
curs in nature and the ability to manipulate 
the organism in the laboratory will open path- 
ways to the genetic study of virulence and 
antigenic variability. 

Histoplasmosis 

Various lines of study have been pursued. 
Several nitrogenous suppdements to sterile soil 
enhanced the multiplication of H. capsulatum 
but in nonsterile soil inhibited growth. These 
studies along with a number of analyses of 
soils from other sites suggest that no single 
nutritional factor determines wheter H. cap- 
sulatum can grow at a particular site. Rather, 
there is indication that the distribution of bac- 
terial antagonists is the limiting factor. 

At the Middle America Research Unit, the 
role of bats in dissemination of histoplasmosis 
was further identified. An outbreak in persons 
following a visit to a bat cave was intensively 
studied with development of both clinical and 
serologic evidence that previous disease was 
relatively protective. The disease was also 
identified for the first time in Western Texas 
when found in free-tail bats. 

LABORATORY OF BIOLOGY OF VIRUSES 

Previously established experimental studies 
on the structure of viral nucleic acids and the 
molecular events involved in virus replication 
have continued during this fiscal year, but, in 
addition, there has been an increased effort 
aimed at determining the functional aspects of 
these nucleic acids. As in the recent past, these 
basic biochemical investigations have not been 
limited to phenomena occurring in virus-in- 
fected cells but of necessity have tested similar 
parameters in normal control cells. 



186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Viral Structure 

Findings on virus structure have varied 
from electron microscopic demonstration that 
polyoma virus may be made up of complex 
lamination of protein layers with a DNA core 
to the characterization of the physiochemical 
configuration of viral RNA and DNA mole- 
cules. Three projects have looked at the struc- 
ture of the nucleic acid from DNA viruses 
and two from RNA viruses. In both types of 
viruses the studies have included single and 
double-stranded forms of the two kinds of nu- 
cleic acids. In fact, in the case of the rat virus 
this work represents the first definitive demon- 
stration that there exists a mammalian virus 
containing single-stranded DNA. A very inter- 
esting "satellite" virus which is a small agent 
unable to replicate by itself and found multi- 
plying only in the same cell with adenoviruses 
has been shown to be a DNA virus whose nu- 
cleic acid is double-stranded. Further infor- 
mation on the structure of the DNA of SV 40 
virus — a tumor inducing agent — has been ob- 
tained through studying the effect of radiation 
on its physical properties. 

Cell Response to Viral Infection 

The examination of the biochemical events 
taking place in the virus infected cell are in- 
separable from study of nucleic acid functions 
of both the cell and the virus. Since messenger 
RNA is required for the production of all spe- 
cific proteins and this function occurs in a 
physical unit made of ribosomes, one of the 
staff has been developing methods for isolating 
and purifying these cellular elements. The im- 
portance of the cellular polyribosomes for 
translating the virus information into viral 
protein products has been demonstrated in the 
vaccinia (DNA), Reo virus (RNA) and polio- 
virus (RNA) systems. Furthermore, in basic 
studies on the mode of action of the impor- 
ant antiviral substance, interferon, evidence 
has been obtained that the effect may be local- 
ized at the level of association of viral RNA 
with ribosomes. 

Viral Oncogenesis 

Polyoma virus DNA synthesis in lytic infec- 
tion has been found to be preceded by the 



early appearance of specific "tumor" antigen 
and an increased activity of enzymes required 
for DNA synthesis. Of special significance is 
the simultaneous increase in cell DNA synthe- 
sis since this may be a requirement for inte- 
gration of viral genome into cell genome during 
oncogenic transformation. Another experimen- 
tal finding having significance concerning the 
relationship of tumor virus DNA to cell DNA 
is the demonstration that a single cell can be 
transformed by two different DNA tumor vi- 
ruses — SV 40 and polyoma. Such doubly trans- 
formed cells contain two types of specific 
tumor antigens induced by both viruses and 
appear to have an increased growth potential. 
This finding raises the possibility that more 
than one site for integration of viral genome 
is available on the genome of the cell. 

Genetic Relatedness 

Several of the laboratory's projects have 
been aimed at nucleic acid functions in the nor- 
mal mammalian cell. Previous results had 
shown a degree of common structure in the 
DNAs of various animal species varying ac- 
cording to their evolutionary relatedness. Now 
by study of pieces of the cell genome, the com- 
monness has been shown to reside chiefly in 
those areas of the DNA highest in G-C base 
content. By manipulation of conditions during 
comparison of, for instance, the DNAs of man 
and chimpanzees, previously undemonstrable 
differences can now be shown. Another signifi- 
cant finding is evidence pointing to the exist- 
ence of repeating sequences of bases in cell 
DNA and the separation of these stretches of 
the genome into classes according to their fre- 
quency of occurrence. Preliminary experiments 
have been initiated on the separation and 
characterization of chromosomes from mam- 
malian cells. Already techniques have been de- 
veloped for obtaining large, workable, amounts 
of partially purified human chromosomes which 
can now be tested for a variety of biochemical 
functions. 

LABORATORY OF TROPICAL VIROLOGY 

Major efforts continued to be related to the 
study of the properties of South American 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



187 



hemorrhagic fever viruses. Amapari virus, a 
new member of the Tacaribe group, was shown 
to be more adaptable to suckling hamsters than 
to suckling mice. The advantages of tissue cul- 
ture cell lines derived from newborn rabbits 
MA III) and green monkey kidney (Vero) 
were investigated for propagation of Tacaribe, 
Amapari and Junin viruses. Each cell line had 
definite advantages and disadvantages. Efforts 
to obtain cytopathogenic effects with Machupo 
virus in human embryonic kidney cell lines 
were essentially unrewarding. 

MIDDLE AMERICA RESEARCH UNIT 
(MARU) PANAMA CANAL ZONE 

Efforts were concentrated toward the fur- 
ther definition of South American hemorrhagic 
fever viruses and on the prevalence of arbovi- 
ruses in Central America. More than 11,000 
sera have been placed on file for screening for 
antibody to arboviruses. The bulk of these sera 
were obtained in collaborative studies between 
MARU and INCAP in Costa Rica during 
ICNND sponsored nutrition surveys through- 
out Central American countries. IBM data 
processing has been utilized for data record- 
ing, retrieval, and analysis of the information 
which is accumulating on arbovirus infections. 

Hemorrhagic Fever 

Continued surveillance in San Joaquin re- 
sulted in diagnosis of 18 sporadic cases of dis- 
where trapping results indicated residual pop- 
ease during calendar year 1965. Most of these 
were concentrated in two areas of the town 
ulations of Calomys callosus. 

Field studies in Bolivia, Peru, Paraguay, and 
Brazil were successfully completed. Testing of 
human sera to date reveals no evidence of past 
HF infection. At least 5 candidate HF agents 
have been recovered from Calomys captured 
in Brazil and San Ignacio, Bolivia. 

Studies in experimental animals were ex- 
tended. Adult Rattus rattus, Proechimysguy- 
annensis and possibly Mus musculus do not 
develop chronic infections with viruria follow- 
ing Machupo virus inoculation. Other rodent 
species are being tested. 



Asymptomatic infection was induced in new- 
born Calomys by Junin, Tacaribe, and Ama- 
pari viruses. Antibodies were produced by all 
viruses. Analysis of animal tissues for evi- 
dence of chronic infection is in progress. 

Cross challenge tests in marmosets indicate 
that infection with Junin or Tacaribe viruses 
does not protect against subsequent lethal Ma- 
chupo infection. Attempts to attenuate Machu- 
po virus for this host are under way. 

Arbovirus Studies 

Continuous sentinel monitoring of virus ac- 
tivity was maintained at Gamboa in the Canal 
Zone. Eighteen strains of VEE virus, seven of 
EEE and one of Bussaquara were recovered 
between June and December 1965. As in pre- 
vious years activity declined below detectable 
levels from January through April. 

A study of dynamics of infection by VEE 
and related Pixuna and Mucambo viruses in 
reservoir rodents Proechimys semispinostis and 
Sigmodon hispidus was completed. Viremias 
ranged from 2-5 days duration, most animals 
survived, and virus was recovered from throat 
swabs, but not feces or urine. Animals devel- 
oped anitbodies to all three viruses following 
infection by any one of them. VEE infection 
conferred complete protection against Pixuna 
and Macambo viruses, but Pixuna infection 
failed to protect completely against subsequent 
VEE challenge. 

A study of antigenic variation among VEE 
strains was begun. It appears that donor host 
is not a significant variable, and that clear 
cut geographic differences are recognizable. 

LABORATORY OF GERMFREE ANIMAL 
RESEARCH 

The laboratory continues to take advantage 
of the germfree animal to study certain infec- 
tious processes and immune reactions under 
conditions where previous infection and im- 
mune responses have not altered the host's re- 
activity. The germ free animal has seemed a 
particularly promising model for the study of 
autoimmune phenomena. 



188 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Immunoglobulins in Germfree Mice 

It has been shown that germfree mice usu- 
ally synthesize and contain in their sera only 
7Syiand IgM globulins. These proteins are 
synthesized by spleen and lymph nodes. In con- 
ventional mice similar tissues form all immu- 
noglobulins. The ileum of conventional mice 
forms principally IgA globulin while that of 
germfree mice usually forms none. Levels of 
7Syi and IgM globulins can be increased by 
immunization with ferritin and the somatic 
lipopolysaccharide of Escherichia coli. A sin- 
gle high dose of endotoxin and two appropri- 
ately spaced, but not a single injection, of fer- 
ritin result in the synthesis of 7Sy 2 globulin 
by the spleen. Formation of this protein shows 
a high correlation with the appearance of ger- 
minal centers in the spleen.The response to 
endotoxin is characterized by a prolonged in- 
crease in IgM globulin to levels between 1.5 
and 2 times control levels, and this response 
is indepdendent of dose between 0.5 and 50 ^g. 
A single injection of ferritin produces a tran- 
sient increase of IgM globulin to 0.2-0.2 mg/ 
ml, about double the control levels, followed by 
a 30 percent increase in 7Sy x globulin. The 
secondary response to ferritin shows an in- 
crease in IgM globulin of a magnitude similar 
to that seen in the primary response, but the 
elevation is much more prolonged. In addition, 
the serum concentration of 7Syx globulin ap- 
proximately doubles. Much of the relatively 
large increases produced by these immuniza- 
tions cannot be accounted for as specific anti- 
body. 

Genetic Factors May Regulate Immune Tissue 
Destruction 

The incidence of autoallergic thyroiditis in 
two strains of guinea pigs was compared. The 
Hartley strain animal developed the disease 
more frequently and after lower antigenic im- 
munizing doses than did the Strain 2 guinea 
pig. Nevertheless, the antithyroid antibody 
titer and delayed skin reactivity in the two 
strains of guinea pigs were identical. These 
strain differences, in the face of an apparently 
equal gross immune response, may be due to 
quantitative differences in the classes of im 



munoglobulins produced, to variations in cell 
structure in which one type may be more easily 
damaged than the other, or to different amounts 
of reactive intermediates which are involved 
in immune tissue destruction. 

Clostridium Perfringens Lethal to Germfree 
Guinea Pigs 

A high mortality rate is associated with the 
transfer of germfree guinea pigs to an open 
animal room where they come into contact 
with a variety of bacterial species. In studies 
designed to investigate the nature of this in- 
fectious process, a strain of Clostridium 'per- 
fringens was isolated from the intestinal con- 
tents of moribund ex-germfree guinea pigs. In 
vitro cultivation of this organism led to the 
production of heat-labile exotoxin (s) which 
on intraperitoneal injection into mice produred 
death. Oral inoculations of germfree\ guinea 
pigs with this Clostridium produced death 
within 12-18 hours. Other investigations have 
shown that conventional guinea pigs often har- 
bor this organism in the intestinal tract yet 
are tolerant to heavy oral doses. These studies 
have provided new knowledge as to the nature 
of this increased susceptibility of germfree 
guinea pigs to bacterial exotoxins. 

Severity of Amoebic Infections Influenced by 
Associated Bacteria 

Conventional guinea pigs harboring a vari- 
ety of intestinal bacterial species often develop 
fatal amoebiasis after intracecal inoculation 
with Endamoeba histolytica whereas germfree 
guinea pigs do not develop severe disease. By 
monocontaminating groups of germfree ani- 
mals with known species of bacteria and in- 
fecting each group with the same strain of 
E. histolytica, differences in severity of the 
infections have been observed depending upon 
the bacterial species present in the intestine. 
When Bacillus subtilis was used as the bacterial 
associate, all animals succumbed to the infec- 
tion. However, the infection of guinea pigs 
with amoebae and a species of Micrococcus re- 
sulted in no mortality. Various gradations of 
severity were observed with other known spe- 
cies of bacteria and, in addition, differences 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



189 



were observed in the intestinal lesions pro- 
duced. These preliminary observations sug- 
gest that the severity of experimental intesti- 
nal amoebiasis in guinea pigs may be influ- 
enced by the species of associated bacteria. 

Low Serum Lysozyme Levels in Germfree and 
Ex-Germfree Rats 

Levels of serum lysozyme in germfree and 
ex-germfree Fisher and Sprague-Dawley rats 
have been found to be significantly lower than 
in conventional animals of the same strains. 
However, no significant difference in enzyme 
free rats. Large amounts of lysozyme are pres- 
ent in leukocytes and a close correlation is 
known to exist between enzyme levels and total 
granulocyte counts. In the three animal groups 
studied, this granulocyte-lysozyme association 
has confirmed, i.e., the lowest levels of lysozyme 
and the lowest leukocyte counts being found 
in the germfree rats. 

Immunoglobulins Quantitated in Human 
Malaria 

The importance of the increases of the var- 
ious immunoglobulin classes formed in re- 
sponse to infection with the malaria Plasmodia 
has been emphazied by recent studies. Serial 
quantitation of the three immunoglobulins 
IgM, IgG and IgA during primary attacks of 
vivax and cynomolgi malaria have shown that 
a close association exists between increased 
immunoglobulin levels and the formation of 
specific malarial antibody. Large amounts of 
IgM and IgG globulin were formed during the 
course of the primary attack in all volunteers 
— the increase in IgA globulin was less strik- 
ing. Volunteers with the milder cynomolgi in- 
fections of lower parasitemia synthesized less 
of the three immunoglobulins than did those 
with vivax malaria. No significant increases 
were observed in IgD globulin during the pri- 
mary malarial infection. Fractionation of sera 
taken from a single vivax volunteer during the 
primary attack demonstrated that both the 
19S and 7S fractions had specific antibody 
against the infecting strain. However, during 
the secondary response, when the patient re- 
lapsed, there was no specific malarial anti- 



body activity in the 19S fraction and antibody 
activity appeared to be confined to the 7S 
fraction. 

Laboratory of Parasitic Diseases 

A relatively broad approach to the problems 
of parasitism has been the primary concern 
of the laboratory, emphasizing basic research, 
coupled with field studies in various areas of 
the world. 

Five field projects have been conducted out- 
side the U.S. Dr. Allen W. Cheever has con- 
tinued his studies on schistosomiasis pathology 
in Salvador, Bahia, Brazil. Two other programs 
on schistosomiasis will reach completion soon; 
one supported by PL-480 funds in Egypt on 
mass treatment with sodium antimony dimer- 
capto succinate (Astiban); the other in Puerto 
Rico on continuous drug infusion. Two addi- 
tional PL-480 programs, one concerned with 
amebiasis in India, the other with toxoplas- 
mosis in Israel, have continued at a productive 
level. 

Schistosomiasis 

The release of cercariae from the snail host 
is not influenced by an innate circadian 
rhythm. Light can stimulate their release in 
the absence of temperature change. If the light 
intensity is at least 60 foot candle power, even 
a few seconds of light can stimulate cercariae 
release. In the absence of light, a temperature 
rise of 4° C. causes their release, but a 2° rise 
does not. 

Low temperatures inactivate cercariae; 
their sedimentation rate at 1-3° C. is 4.95 ± 
0.09 cm per minute. At 11 to 13° C. cercariae 
become sufficiently inactive so that they cannot 
maintain their positional level in the water; 
these data suggest that human infection will 
not occur in water below this temperature 
range. 

The determination of cercarial concentra- 
tions in natural bodies of water is fundamental 
to an understanding of their population dy- 
namics and to the transmission of the infec- 
tion. A simple continuous flow centrifuge has 
been developed for the quantitative collection 
of cercariae. Seventy to ninety percent of the 



190 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



organisms were consistently recovered from 
one-liter water samples and over 50 percent 
from larger samples. The technique appears 
promising for field use. 

Laboratory studies indicate that miracidia 
infected snails 10 meters from their point of 
release, and were infective up to 8 hours after 
they hatched. These and other data suggest 
that miracidia have a capacity to infect snails 
in the field at considerable distances from 
their point of release from the schistosome egg. 

Highly important findings were made con- 
cerning the pathogenesis of huma schistosome 
infection based on quantitative data obtained 
from perfusion of cases autopsied at the Hos- 
pital Professor Edgard Santos in Bahia, Brazil. 
The study has revealed a number of parasito- 
logic differences between patients with and 
without disease, i.e., with or without Symmer's 
fibrosis. Patients with Symmer's fibrosis often 
have heavy infections, and many of those with 
lower worm burdens have definite or presump- 
tive evidence of previous treatment. Egg dis- 
tribution is distinctive in cases of Symmer's 
fibrosis. The majority are found in the small 
intestine, and rectal tissues contain very few 
eggs. The opposite is true in cases without evi- 
dent anatomic disease. In Symmer's cases, eggs 
in the liver are concentrated in the portal 
areas, whereas in the other cases they are dis- 
tributed randomly within the liver. Approxi- 
mately 8% of "asymptomatic" cases have 
worm burdens comparable to those of the more 
heavily infected Symmer's cases. The factors 
determining the presence or absence of dis- 
ease are not clear. 

Collaborative studies on the effect of single 
monthly injections of Astiban to village popu- 
lations in Egypt were continued. These stud- 
ies have shown that urinary shedding of ova 
of S. hematobium ceased in about 90 percent 
of recipients under such therapy and had not 
resumed in any significant number of victims 
during a six month period after therapy was 
stopped. These observations indicate that the 
drug in this dosage has both chemoprophylac- 
tic and chemotherapeutic properties and the 
method would seem to be suitable for mass 
treatment of populations in hyperendemic 
areas. 



In Puerto Rico it was shown that a slow 
continuous infusion or a series of closely spaced 
small injections of Stibophen was better tol- 
erated by the patient and therapeutically more 
effective than were larger, more widely spaced 
injections. Very similar observations were 
made with Astiban (sodium antimony dimer- 
capto succinate). 

Amebiasis and Trichomoniasis 

With the aid of a sensitive microfluorimeter 
and measuring techniques developed in this 
laboratory, it has been possible for the first 
time to demonstrate a spectrum of antigenicity 
in some 16 strains or lines of Entamoeba his- 
tolytica growing in association with a mixed 
bacterial flora. It has also been shown that 
antigenic differences exist between 3 strains 
of Trichomonas, two of them derived from a 
single source, but maintained under different 
conditions for 18 months. 

Fluorescent antibody serology has confirmed 
a high incidence of amebic liver abcess in pa- 
tients seen at the hospital in Hyderabad, India. 
In addition, about 55% of cases with the vague 
symptomatology of chronic intestinal or he- 
patic amebiasis" are positive in the FA test 
for amebiasis, compared to 30% of patients 
without such clinical signs. There is consider- 
able serologic evidence accumulating that the 
amebiasis picture in countries such as the 
United States cannot be compared in any real- 
istic manner to that in an endemic area such 
as India. There has been no correlation be- 
tween positive serologies and infection with 
E. hartmanni, so-called "small race E. histo- 
lytica," thus supporting the view that this is 
a separate species from E. histolytica. 

Continued improvement has been made in 
the medium used for the axenic cultivation of 
E. histolytica. Considerable variation was en- 
countered in the ability of different lots or 
horse serum and liver extract, respectively, to 
support growth. These axenic cultures have for 
the first time made available a system for the 
testing of drugs directly against amoebae. Prior 
tests conducted on amoebae grown in associa- 
tion with microbial flora were always incon- 
clusive because of the possibility that the drug 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



191 



acted on the microbes which served as food 
for the amoebae. Humatin (Paromomycin sul- 
fate) killed axenic amoebae at levels of 8 and 
16 micrograms/ml medium. 

In vitro synthesis of polyamino acids was 
demonstrated using - subcellular fractions of 
axenic E. histolytica under controlled condi- 
tions. Tetracycline, an antibiotic apparently 
without an effect on the mammalian protein 
syntehtic machinery, was markedly inhibitory. 
This provides the first direct evidence that the 
therapeutic action of tetracycline in amebiasis 
is specifically on the amebae and not on the 
associated bacteria, and affords a clue as to its 
mechanism of action. Polyribosomes from the 
amoebae, constitute the first identification of 
these biochemical structures in a protozoan 
cell. 

Glucose and Glycerol Utilization in Parasites 

Comparative studies of glucose and glycerol 
uptake by the larval and adult forms of Taenia 
teaeniaeformis, and by different species of try- 
panosomes under various conditions have re- 
vealed interesting similarities and differences. 
The larva absorbs more glucerol aerobically 
than anaerobically, a difference lacking in the 
adult worm. For the larva, an inverse relation 
exists between concentration of environmental 
glycerol and glucose leakage. In the absence 
of Na + , marked inhibition of glucerol and glu- 
cose uptake occurs, the effect being more pro- 
nounced on the latter. Phloridzin completely 
inhibits glucose uptake, but has no effect on 
that of glycerol. The data suggest that at very 
low environmental glycerol concentrations, ab- 
sorption involves active transport mechanisms, 
while at higher concentrations diffusion, prob- 
ably facilitated diffusion, appears to prevail. 

Of 8 species of trypanosomes studied, only 
T. gambiense and T. rhodesiense consumed 
large amounts of glucerol. When T. gambiense 
was kept in a mixture containing equal 
amounts of glycerol and glucose, definite mu- 
tual inhibition of absorption was observed. In 
addition, other differences between tapeworms 
and T. gambiense were found to exist in respect 
to extraneous influences. 



Laboratory of Parasite Chemotherapy 

The major research effort of the laboratory 
continued to be centered on problems in ma- 
laria. Resistance of strains of falciparum ma- 
laria to all commonly used synthetic antima- 
larial compounds remains a severe problem, 
particularly in Vietnam and other areas of 
Southeast Asia. The need for new and effec- 
tive drug regimens to combat this problem has 
dictated continuation and expansion of this 
area of research. Further studies on the many 
host-parasite-vector combinations now avail- 
able in the simian malarias have contributed 
much to our program of evaluation of antima- 
larial agents and to our understanding of the 
biology of the parasite in the primate host. 

Malaria — Human 

Studies continued in the clinical testing of 
antimalarial drugs in human volunteers, with 
emphasis on strains of parasites resistant to 
certain compounds and on possible methods for 
overcoming the resistance problem. Activity of 
cycloguanil pamoate (CI-501), the injectable 
long-acting antimalarial, and that of CI-564, 
a combination of CI-501 and DADDS (the 
acetylated diaminodiphenyl sulfone), was eval- 
uated against strains of vivax and falciparum 
malarias resistant to chlorguanide, the parent 
compound of CI-501. While CI-564 was effec- 
tive against the normal strain of Plasmodium 
vivax, when the strain had been made chlor- 
guanide-resistant the drug had only minimal 
antimalarial activity. Trails of CI-564 against 
multiresistant strains of P. falcipamm were 
encouraging. Although these strains were re- 
sistant to normal doses of chlorguanide or 
CI-501, in most cases volunteers were suc- 
cess fully protected from or cleared of infection 
when CI-564 was used as a suppressive or 
therapeutic agent. 

Four additional strains of drug-resistant 
falciparum malaria were isolated and studied 
in volunteers. These strains were all from 
Southeast Asia and included one from Thai- 
land (Thai II), one from Malaysia (Malayan 
IV) and two from South Vietnam (SV-1 and 
SV-II). The first three strains have been ex- 
tensively evaluated and all possess a wide spec- 



192 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



trum of resistance, being refractory to chloro- 
quine, chloroguanide and mepacrine. The SV- 
I strain appeared to be sensitive to 50 mg. of 
pyrimethamine, but the others were also re- 
sistant to this compound. While quinine sulfate 
was curative when given as a 5-day course of 
treatment in cases of Thai II and Malayan IV, 
THE SV-I strain appears to be resistant to a 
10-day course of quinine, requiring 14 days of 
this drug for a cure. The SV-II strain is a more 
recent isolate, and was derived from an infec- 
tion which had recrudesced after extensive 
therapy with quinine (10 grains t.i.d. X 14 
days, followed by 5 grains t.i.d. for 6 weeks). 
This strain is resistant to chloroquine and 
chlorguanide. 

In line with the urgent need for alternative 
methods of treatment of the resistant strains, 
studies have been done on the usefulness of 
sulfonamides, either alone or in combination 
with pyrimethamine. Forty volunteers have 
participated in the evaluation of thee sulfon- 
amides: sulfadiazine, sulfamethoxypyridazine 
(Midicel), and sulforthodimethoxine Fana- 
sil). The Malayan III strain of P. falciparum 
was used for the initial studies; it is resistant 
to all synthetic antimalarials tested, but sen- 
sitive to the 5-day treatment with quinine sul- 
fate. Sulfadiazine was given in a 5-day course 
of treatment, while the longer acting com- 
pounds. (Midicel and Franasil) were given as 
single doses. Results indicated varying degrees 
of antimalarial activity for all three of the 
sulfonamides when used alone, but that none 
was completely reliable for the termination of 
these malarias. When pyrimethamine (as a sin- 
gle dose) was given concurrently with the 
sulfonamides the curative effect was signifi- 
cantly enhanced. A pyrimethamine-Fanasil 
combination was evaluated against three addi- 
tional resistant strains: Thai II, Malayan IV 
and South Vietnam I. In a total of 24 volun- 
teers treated, this combination was successful 
in effecting a cure in 21. 

Studies have continued on the West African 
strain of P. ovale to characterize the clinical 
and parasitological patterns of this infection 
in volunteers. Contrary to prior descriptions, 
ovale malaria appears to present moderately 
severe clinical manifestations. The strain re- 



sponds readily to standard antimalarial regi- 
mens. A single dose of 10 grains of quinine 
sulfate successfully clears patent paraistemias 
in about four days. Observations on relapse 
patterns of quinine-treated, mosquito-inocu- 
lated cases indicate a wide range of intervals 
between initial and subsequent attacks. While 
relapse has been seen as early as 17 days after 
treatment of the primary attack, of particular 
interest are the extended latent periods some- 
times seen. In one case the interval between 
primary attack and first relapse was 255 days. 

Malaria — Simian 

Studies on simian malaria as a possible zo- 
onotic disease of man have been conducted on 
several fronts. A new strain of Plasmodium 
brasilianum, the South American quartan par- 
asite of monkeys, has Deen transmitted to vol- 
unteers through the bites of infected mosqui- 
toes. The infectionin man was extremely mild 
toes. The infection in man was extremely mild, 
man to man through subinoculation of infected 
blood. Similarly, the quartan parasite of mon- 
keys of Southeast Asia, P. inui, has been suc- 
cessfully transmitted to volunteers through the 
bites of infected mosquitoes. Clinical manifes- 
tations were mild. The infections were self- 
limiting and antimalarial intervention not re- 
quired. Similar exposure of volunteers to mos- 
quitoes infected with P. gonderi, P. Coatneyi, 
P. fieldi, and several additional strains of P. 
cynomolgi have not resulted in detectable par- 
asitemias. 

Of great interest has been the isolation of a 
strain of P. knowlesi from a person recently 
returned from Malaya. This strain has been 
studied in 8 volunteers and in rhesus mon- 
keys. The infection is characterized in man 
by moderately severe clinical manifestations. 
The fever pattern is quotidian, with fever as 
high as 104.8° F. The maximum parasite count 
has been about 20,000 per cmm of blood. The 
infection is generally self-limiting, but anti- 
malarial intervention has been deemed advis- 
able in three cases. It has been possible to 
infect monkeys through mosquitoes fed on 
human carriers. The course of infection in 
monkeys is generally very severe, almost in- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



193 



variably terminating- fatally in the absence of 
antimalarial intervention. This is the first doc- 
umented case of a human acquiring a monkey 
malaria under natural conditions and definitely 
establishes it as at least an occasional zoonosis. 
To follow this lead, studies were re-established 
in Malaysia, particularly in the area from 
which this case was known to have originated, 
to determine the extent to which this zoonosis 
might exist as a real problem. Results of this 
limited study are not yet complete; however, 
it seems evident that, although this zoonosis 
may occur occasionally, it is not a significant 
problem in the overall consideration of malaria 
in these areas at this point in time. 

Extensive studies on the exoerythrocytic 
stages of the simian malarias have been initi- 
ated, primarily to provide a reliable system 
for the evaluation of antimalarial compounds 
against these cryptic forms, but also to expand 
our knowledge of the entire life cycle of the 
primate malarias. Techniques have been de- 
vised for the direct massive intrahepatic inocu- 
lation of sporozoites, which enhances the loca- 
tion and identification of these stages on sub- 
sequent biopsy. Using this method it has been 
possible to demonstrate developmental exoery- 
throcytic stages of P. coatneyi, P. fieldi, P. 
knowlesie, P. brasilianum and several strains 
each of P. inui and P. cynomolgi. Preliminary 
studies are under way to determine the activ- 
ity of known antimalarial agents against these 
forms. 

Immunological Studies 

The availability of a large number of human, 
simian, avian and rodent malarias has made 
possible continuation of studies on the devel- 
opment and persistance of specific antibody, 
as determined by the fluorescent antibody 
(FA) method and methods of Immunoelectro- 
phoresis. Monkeys infected with various spe- 
cies of malaria normally develop antibody re- 
sponse to a very high level within three weeks 
after infection. These levels seldom increase, 
even though the parasitemia persists for ex- 
tended periods. 

FA tests have been conducted on several 
large series of serum samples from Nigeria, 



Upper Volta and Liberia (West Africa). These 
era were tested against five Plasmodium anti- 
gens and the responses, in general, indicate an 
increase in titer with age and higher titers in 
males than infemales. In all groups under 13 
years of age, the highest response was to the 
P. falciparum antigen. Those over 13 years of 
age had equal mean titers to the P. falciparum 
and the P. brasilianium antigens, the latter 
species being an indicator of previous P. ma- 
la riae infection. 

Examination of sera from individuals in- 
fected more than 10 years previously with P. 
vivax (during the renowned Campfire Girls 
epidemic in California) indicated that, although 
the FA responses were weak, they could be 
distinguished in most cases from persons of 
the same age who had not experienced malaria. 

Antigenic analyses of the simian malarias, 
are being done preliminary to use of parasiten 
or their components in experiments on immu- 
nization. Electrophoretic analysis reveals a 
minimum of four or five distinct components. 
Immunoglobulin fractions have been collected 
from 10 strains or species and are being used, 
along with whole parasite extracts, for immu- 
nization of animals. 

LABORATORY OF IMMUNOLOGY 

Autoimmunity 

Immunofluorescence studies have established 
that serum immunoglobulins from patients 
with myasthenia gravis react in vitro with 
striated muscle of a wide variety of both ver- 
tebrate and no n- vertebrate species. The occur- 
rence of striated cells in the thymus of several 
vertebrate species has been confirmed; recip- 
rocal absorption studies with striated muscle 
and thymus have suggested common antigenic 
determinants in these tissues. By electron mi- 
croscopy it was established that there are 
thymic cells possessing the sarcomeric and 
myofilamentous structure of striated muscle. In 
collaboration with NINDB it was shown that 
the occurrence of these immunoglobulins in 
human sera is correlated with the occurrence 
of thymomas but that in the thymomas 
studied, there was no correlation with my- 
asthenia gravis. 



194 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



Thyroid gland transplants have been made 
into deep intramuscular sites in inbred Strain 
13 guinea pigs. A significant number of the 
Strain 13 isografts of normal thyroids showed 
evidence of mononuclear cell infiltrates some 
weeks later. This partial rejection of normal 
thyroid isografts constitutes an indication that 
skin grafts may not be a wholly adequate in- 
dicator of histocompatibility of other tissues 
or organs. Some of the findings on lymph node 
transfers given in a previous annual report 
have also been consistent with such a possi- 
bility. 

In view of the age and familial influences 
on certain human demyelinating diseases, and 
prior experiments on experimental autoim- 
mune encephalomyelitis (EAE) in guinea pigs, 
studies on EAE were extended to primates. 
Guinea pig spinal cord in complete Freund's 
adjuvant was administered to Rhesus monk- 
eys of various ages as a single injection. Ani- 
mals ranging in age from laboratory-bred new- 
borns, infants, and juveniles to fully mature 
adults proved susceptible to allergic encephalo- 
myelitis. Of 22 animals tested, all developed 
marked neurological disorders and only two 
survived the disease. Severe inflammatory les- 
ions were found in the central nervous system. 
Hemorrhagic retinopathy preceded or accom- 
panied the clinical and neurological symptoms 
in nearly all cases. Since in neonates and pre- 
matures the onset of clinical manifestations 
was delayed and took a more prolonged course 
and the pattern and distribution of brain 
lesions differed between newborn and older 
animals, there is an age-dependent factor in 
this disease in monkeys. 

Transplantation Immunology 

Pretreatment of donor mice with any of 
wide variety of antigens apparently unrelated 
to transplantation antigens leads to markedly 
diminished capacity of donor lymphoid cells 
to evoke in recipient mice fatal graft-vs-host 
reactions across H-2 histocompatibility bar- 
riers. Alterations in donor spleen cells were 
not a reflection of coexistent lymphoid hyper- 
plasia induced by some antigens, rather re- 
duced donor cell immunocompetence appeared 
to be related to some form of antigenic com- 



petition. Pretreatment of donors with a single 
antigen by no means pre-empted the available 
population of competent cells. Instead, it ap- 
pears to impose an overall reduction in their 
capacity to respond to a second unrelated an- 
tigenic stimulus. This kind of modification of 
donor immunocompetence represents a novel, 
unrestricted approach to the development of 
transplantation tolerance in higher mammals. 

Hypersensitivity 

The availability of inbred strains of guinea 
pigs, a species in which the different facets of 
the immune response are best known, has been 
utilized as a unique resource for analysis of 
the mechanisms and genetics of hypersensi- 
tivity. Use of chemically defined synthetic 
polypeptides for this purpose is advantageous 
in that the specificity of the resulting immune 
response is assured by the known chemical 
composition and the hereditarily determined 
capacity to respond against these structures. 
The presence of lysine residues in otherwise 
antigenic copolymer synthetic polypeptides con- 
sistently results in the lack of antigenicity for 
Strain 13 guinea pigs; immune response in 
Strain 2 pigs is normal. These findings open 
the way for investigation of issues such as 
whether a peptide need be antigenic in a given 
host in order to evoke tolerance and to affect 
the response to another antigen. 

Mitogens 

The plant mitogens from the red kidney bean 
Phaseolus vulgaris and from the root of the 
pokeweed Phytolacca americanus, each of 
which produce distinctive immunologic and bi- 
ochemical transformation of human peripheral 
lymphocytes, have been progressively purified 
and partially characterized. Saline extracts of 
these source materials display hemagglutinat- 
ing, leukagglutinating and mitogenic proper- 
ties. Mitogenic activity was found by different 
analytical methods to be associated with a sin- 
gle protein component. Purified mitogens 
proved to be glycoproteins — but displaying 
different biological characteristics. Distinctive 
chromatographic and electrophoretic mobilities 
for the kidney bean and the pokeweed mito- 
gens were indicative of separate and distinc- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



195 



tive structures. The transformation in vitro of 
peripheral blood lymphocytes by the pokeweed 
mitogens involves two transformed cell types: 
a large blast-like cell indistinguishable from 
that seen in in vitro transformation by the 
kidney bean mitogen and cell type at present 
seen in mitogenic transformation, with fea- 
tures resembling early antibody-forming cells. 
Histcqhemical and radioautographic studies 
on pokeweed-stimulated cultures revealed that 
50-60 percent of the initial population of cells 
were transformed by the pokeweed mitogen 
and that prominent among these was a dis- 
tinctive cell type with cytologic characteris- 
tics resembling plasma cells. 

ROCKY MOUNTAIN LABORATORY 
HAMILTON, MONT. 

Selected Zoonoses of Regional Importance 

Rabies was isolated from five bats, four of 
which were collected in western Montana and 
one in Idaho. One isolate is particularly note- 
worthy since it was obtained from a sick bat 
collected in December. 

The discovery that mice frequently survive 
after rabies infection prompted an inquiry into 
the role of maternal antibodies on the resis- 
tance of offspring of immune white mice. 
Placental transfer of antibodies could not be 
shown, but lacteal secretions were found to 
account for acquired resistance and colostrum 
was not more important than milk. Immun- 
ity, which increased with continued suckling, 
existed for more than 35 days after birth. 
In other studies on immunity against rabies 
attempts were made to characterize a rabies- 
virus-inhibiting substance which develops in 
brains of animals that have recovered from 
rabies. This virus-neutralizing factor did not 
appear after vaccination, even after live virus 
vaccination. As the titer of the inhibiting sub- 
stance increased, the virus content of the brain 
tissue decreased. The ability to distinguish re- 
covered animals from other immunes by dem- 
onstration of this inhibitory substance enables 
a study of the role of abortive rabies in the 
natural epidemiology of the disease. 

Last year it was shown that an ether extract 
of F. tularenis was a reliable skin-test antigen 



for the detection of past infections with this 
organism. This test was applied to laboratory 
employees who had been vaccinated with Ft. 
Detrick live tularemia vaccine. All individuals 
developed agglutinins ranging in titer from 
1:20 to 1:640 but mean titers were lower than 
those expected after natural infection. Most 
vaccinated individuals reacted positively to the 
skin-test antigen but the average size of reac- 
tion was smaller than that arising in people 
who have had the natural disease. 

Rickettsial Diseases 

Investigations on the role of domestic ani- 
mals and wildlife and their ectoparasites on 
the epidemiology of typhus have been contin- 
ued in Egypt and South America. In Egypt 
serologic evidence of infection of livestock was 
found again this year, but serum titers were 
not as high. Among wildlife tested, antibodies 
against Rickettsia proivazekii have been 
shown, with reasonable certainty, in only two 
specimens of Rattus rattus. So far, attempts 
to isolate typhus rickettsiae from 250 wild 
rodents and ticks collected from them or do- 
mestic animals have failed. In South Ameri- 
can studies, supported in part by the Pan Amer- 
ican Health Organization, serologic surveys 
were conducted in two known endemic areas 
in Chile. Typhus antibodies were not detected 
in serums taken from cattle, sheep, goats, 
llamas, or burros, but 12 of 80 human serums 
collected near La Quiaca were positive, three 
of which had dominant antibodies against epi- 
demic typhus. 

In contrast to observations in Egypt and 
Chile, findings in Ethiopia indicate that ty- 
phus is still prevalent. Of 99 serums submitted 
from patients thought to have had a rickett- 
sial infection, none reacted with R. conori, 
but 62 were positive for Q fever and 43 were 
positive for typhus; 19 of the 43 reacted prin- 
cipally against R. proivazekii, 7 against R. 
typhi and the remaining 17 had equal titers 
against both antigens. This year Dr. Reiss- 
Gutfreund submitted three strains of typhus 
to RML for confirmation of their identity. 
These strains, isolated in the same area from 
which previous isolations were made from live- 



196 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



stock and ticks, were identified as follows: R. 
prowazekii (JRS) isolated from Hyalomma 
truncation taken from cattle; R. typhi 
(ZH97) from H. truncatum taken from cattle; 
and R. typhi (L308) isolated from human body 
lice. 

A burro and two goats were inoculated with 
the ZRS strain of R. prowazekii to determine 
the susceptibility of these hosts and to evalu- 
ate the serologic response after infection. At- 
tempts to isolate rickettsiae from the blood of 
these animals failed but all developed higher 
antibody titers against epidemic than against 
endemic typhus. 

Transovarial passage of R. rickettsi in Der- 
macentor andersoni has been investigated in 
ticks fed on normal and spotted-fever-immune 
guinea pigs. For six generations, 12 lines of 
D. andersoni fed on normal nimals trans- 
mitted the virulent R type to 100 percent of 
their progeny. In the same number of genera- 
tions, 8 of 19 lines of D. andersoni fed on 
immune animals lost rickettsial infection. In 
the remaining 11 lines, 100 percent transo- 
varial passage occurred and virulence of the 
R type was not changed. 

Further studies were made on the immuno- 
logic and serologic responses to Q fever vac- 
cines. A single dose of phase I or II vaccine 
in guinea pigs was as effective as two doses, 
provided the same total quantity was given. 
Antibody of the 19S type was produced for 
the first 30 days, after which 7S appeared. A 
dim ethylsulf oxide extract of phase I antigen, 
which is a polysaccharide-fatty-acid complex, 
protected guinea pigs and induced the produc- 
tion of only phase I antibody. 

Field trials with human volunteers of phase 
I and phase II vaccines have been organized 
under the supervision of the AFEB Commis- 
sion on Rickettsial Diseases. The results so far 
indicate that the administration of 30 fig of 
Henzerling phase I vaccine in a single dose 
provided complete protection against 3000 
GPID r „, aerosol doses. Similar results were ob- 
tained when 165 fig on Henzerling phase II 
vaccine was employed, but a four-fold increase 
in phase I agglutinins in three of the six men 
challenged later suggests that limited multi- 



plication of the challenge organisms may have 
occurred. 

Reference examination of serums by the ra- 
dioisotope precipitation (RIP) test is one of 
the services provided by this laboratory as 
WHO Regional Reference Center for Human 
Rickettsioses. Of all serologic tests for Q fever, 
the RIP test is considered to be most sensitive 
and specific. Among various groups tested, in- 
fected rates have varied from 2% to 66%, 
a rate found among veterinarians in large 
animal practice. Clinical Q fever has not been 
a factor in any of the groups studied. In other 
representative tests 4.3% of 208 patients in 
tuberculosis hospitals in the Midwest and 62% 
of 99 persons in Ethiopia, who were thought 
to have had typhus, were seropositive. 

Transmission of Disease Agents by Vectors 

In several collaborative efforts, chiefly with 
NAMRU-3 in Egypt, some major segments of 
classification of ticks from various parts of the 
world were completed. Some important taxo- 
nomic studies of the New World chiggers also 
were undertaken. During the year, 52 new 
species, of which six were assigned to new 
genera, were described in papers published or 
in press. 

Investigations have been continued on the 
genetics of a paralytigenic factor in Derma- 
centor andersoni; the 7th generation of an 
inbred line from British Columbia has shown 
the same remarkable ability to cause tick pa- 
ralysis as did previous generations. In view of 
the suspected relationship of Wolbachia in 
Argas arboreus to this tick's ability to par- 
alyze pigeons, the causal relationship of Wol- 
bachia recently isolated from D. andersoni to 
tick paralysis in mammals is being in- 
vestigated. 

Other research in this project has been 
quite diverse. Studies have been continued on 
three strains of the Hughes virus complex iso- 
lated from Ornithodoros denmarki and O. 
capensis. Eastern equine encephalitis (EEE) 
virus was shown to survive in Ornithonyssits 
bacoti for five to seven days. In other studies 
involving this virus, the common mealworm, 
Tenebrio moiitor, was evaluated as a research 
tool. The larvae, which were found to be as 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



197 



susceptible as mosquitoes, yielded virus titers 
as high as 10 6 when held at 98° F. 

Encephalitides and Tick-Born Diseases 

In view of the reported isolation of WEE 
from naturally infected snakes, studies were 
conducted during an acute outbreak of WEE 
in northeastern Montana to determine whether 
snakes may acquire virus from C. tarsalis-bird 
cycle. Since 7 or 10 pools of C. tarsalis col- 
lected during the outbreak contained WEE 
virus, and 64% of 85 chickens in flocks in the 
immediate area had developed antibodies, the 
outbreak was characterized by intense virus, 
activity. Eighteen cases in man were con- 
firmed serologically and an estimated 300 cases 
occurred in horses. More than 300 snakes of 
five species and 110 leopard frogs were col- 
lected during the post-epidemic period and 
tested under various conditions supposedly con- 
ducive for the isolation of virus, but none was 
recovered. 

Studies were renewed on the immunologic 
response in human volunteers vaccinated with 
an experimental Colorado tick fever vaccine 
in 1961. In March 1965, 7 of 10 individuals 
still had neutralizing antibodies varying in 
index from 32 to as high as 4,470. After skin 
test with the vaccine all 10 persons developed 
a significant rise in titer, persumably from 
the small amount of antigen used in the skin 
test. 

PGittacosis-Lymphogranuloma-Trachoma ( PLT ) 

Research on the PLT group of agents has 
been conducted chiefly to develop and improve 
methods of isolation and purification, to de- 
velop methods whereby more luxuriant growth 
can be obtained, and to develop more sensitive 
and specific diagnostic reagents. Of two new 
cell lines established (mouse embryo and 
mouse placenta) the latter was more suitable 
for the propagation of PLT agents. 

The radioisotope precipitation (RIP) test 
continues to show promise as a highly sensi- 
tive technique for detecting antibodies against 
the PLT group. Several aspects of the test, 
however, require further study before it can be 
used as a practical tool. Since P 32 has a rela- 



tively short half life, a new batch of antigen 
has to be prepared about every two months. 
This difficulty has been partially overcome by 
labeling with C 14 -tagged adenine, but, un- 
fortunately, only a small part of the label was 
incorporated into the meningopneumonitis par- 
ticles. Further studies with other C 11 compon- 
ents are planned to find one with a higher rate 
of radioisotope incorporation. 

Screen tests have been conducted on various 
groups of human serums. Significant titers 
were demonstrated on the serums of 15% of 
415 Montana fur trappers, 25% of 29 Dubois 
sheep station personnel, and 23% of 88 North- 
ern Cheyenne Indians. The RIP test con- 
firmed CF findings in a group of serums from 
14 patients with "chronic carditis," all of 
whom had PLT group CF antibodies. 

The significance of these interesting results 
is as yet unknown. Five of six serums from 
clinical cases of lymphogranuloma venereum 
(LGV) had RIP antibodies. The RIP test ap- 
pears to be more sensitive than the CF test 
in detecting prior PLT infection. These find- 
ings suggest that PLT group infections are 
far more prevalent than they generally were 
thought to be. 

Further epidemiologic study of trachoma in 
two boarding schools on the Northern 
Cheyenne Indian Reservation were conducted 
during the year. In March 1965, 538 students 
were examined — 82 were thought to have 
trachoma. A single isolation was made from 
a student with stage I trachoma. Serums ob- 
tained from these students were tested by both 
the CF test and the RIP test. By the CF 
test, 25% of serums from possible cases had CF 
activity in dilutions of 1:4 or greater. None 
of these serums reacted to trachoma type- 
specific cell- wall antigens. In contrast, 58% of 
these serums reacted positively in the RIP 
test. The results of the RIP test also correlated 
well with the clinical classification of eye 
disease. Serologic tests of serums from a few 
adults with stage II or IV trachoma revealed 
the presence of high RIP titers in the absence 
of any CF activity. This finding suggests that 
the two tests measure different type of anti- 
body and that the RIP antibody response is 
more durable than the CF response. 



198 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Chronic Progressive Viral Disease 

This year another animal disease, chronic 
interstitial pneumonitis of sheep, was shown 
to be caused by a slow-growing filterable 
agent. In the 15 months this project has been 
underway typical disease appeared in four of 
eig'ht sheep inoculated intravenously with fil- 
trates of a suspension of lungs from naturally 
infected sheep. Three of nine sheep which re- 
ceived similar material directly into the right 
lung also developed the disease. Sheep inocu- 
lated intracerebrally with similar material 
have remained healthy as have goats inocu- 
lated either intravenously or directly into the 
lung. The extent of lymphoreticular prolifera- 
tion in the lungs of sheep that died or were 
killed was particularly striking. 

Additional results of an experiment begun 
in August 1962, more clearly indicate the dif- 
ferences in the distribution of scrapie virus 
during advanced disease between goats inocu- 
lated intracerebrally and those inoculated sub- 
cutaneously. In the former, virus was virtually 
limited to the nervous system, whereas in those 
inoculated subcutaneously that became infected 
with scrapie 19 and 21 months after inocula- 
tion, virus was found in many extraneural 
sites, notably lymphocytic tissues. Such wide 
distribution outside the nervous system has 
not been found so far in a goat that did not 
develop disease until 33 months post inocula- 
tion. Results have again indicated the impor- 
tance of lymphocytic tissues in the pathogene- 
sis of scrapie. During the first 12 months after 
inoculation only one of 15 goats yielded virus, 
a small amount in the left prescapular lymph 
node that drained the site of inoculation. Dur- 
ing nine months of the second year, virus was 
detected in six of nine goats examined. Of 36 
tissues regularly tested, only lymphocytic tis- 
sues contained detectable amounts. Twenty- 
nine months after inoculation, signs of clinical 
disease have not yet appeared in 15 remaining 
goats. Results obtained in this experiment are 
interpreted as indicating a long latent period, 
during which virus cannot be detected, fol- 
lowed by slow replication and spread of virus 
primarily in lymphocytic tissues. 

The agent which causes encephalopathy of 
mink was found to pass through both 100 imi 



millipore niters and 100 m^ gradacol mem- 
branes. Thus, the size of the infectious agent 
is well below that of known non-viral patho- 
gens. In Pastel mink severely affected with 
this disease the largest amount of virus was 
found in the brain, with lesser amounts in 
various other tissues, but it could not be recov- 
ered from blood or serum. When small amounts 
of virus were present in the inoculum, incuba- 
tion periods of over 400 days were observed. 
The viremia that occurs in Aleutian disease 
was studied in Sapphire (an Aleutian color 
phase) and Pastel (a non- Aleutian color 
phase) mink. In Sapphire mink the virus could 
be recovered at biweekly intervals until death 
which occurred about 12 to 22 weeks after 
inoculation. During this period the levels of 
serum gamma globulin rose by 40-45% with- 
out any apparent effect on the presence of 
virus in the blood. In Pastel mink virus was 
recovered at two weeks and 12 weeks post 
inoculation but not thereafter through 22 
weeks. Pastel mink remained free of serum 
protein changes and are still healthy 32 weeks 
after inoculation. In another experiment virus 
was detected in the mesenteric lymph nodes of 
two apparently healthy Pastel mink 22 months 
after inoculation. These observations on 
viremia indicate a clear-cut difference in the 
mode of virus growth in the two color phases 
and further emphasizes the importance of 
genotype in the pathogenesis of the disease. 

Allergy and Immunology 

Further comparisons have been made of the 
Hart.ey strain guinea pigs and strain 13 guinea 
pigs regarding their suitability for research 
on delayed hypersensitivity. Previously, strain 
13 guinea pigs were shown to produce anti- 
body less actively than did the Hartley strain. 
Strain 13 guinea pigs developed pure delayed 
hypersensitivity to egg albumin when sensi- 
tized with a conjugate of paraaminobenzoic 
acid (PABA) and hen egg albumin (HEA), 
whereas Hartley strain guinea pigs developed 
an immediate type of hypersensitivity. When 
both strains were inoculated with live BCG, 
they developed hypersensitivity to tuberculin 
but the reaction in strain 13 guinea pigs were 
much weaker. When these guinea pigs were 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



199 



sensitized with killed Q fever phase I ricket- 
tsiae and tested several weeks later with phase 
I cells, reactions were similar in both strains. 

Immunoprophylaxis Against Tuberculosis 

Previous studies have shown conclusively 
that heat-treated cell walls of Mycobacterium 
tuberculosis when coated with light mineral oil 
are highly immunog-enic and protect mice 
against aerosol challenge with fully virulent 
tubercle bacilli. Over a two-year period numer- 
ous vaccines have been prepared from 12 differ- 
ent batches of cell walls and tested repeatedly. 
Except for a few formulated with an insuffi- 
cient quantity of oil, all vaccines have been 
highly potent in repetitive tests. Mice immun- 
ized with these cell-wall vaccines have har- 
bored up to five logs fewer virulent mycobac- 
teria in their lungs after aerosol challenge than 
did unvaccinated control mice. In contrast, 
mice vaccinated with the standard Rosenthal's 
BCG vaccine had about two logs fewer when 
they were cultured after aerosol challenge. 
None of several vegetable oils, which are 
metabolizable, served to enhance immunogen- 
icity of cell-wall preparations. Both heavy and 
light mineral oil were effective whereas kero- 
sene was ineffective in enhancing potency. 
Drakeol 6VR, which has been approved as an 
adjuvant in human vaccines, was found to be 
as satisfactory as light mineral oil. The syn- 
thetic hydrocarbon, 7-n-hexyloctadecane, was 
slightly superior to light mineral oil. The 7-n- 
hexyloctadecane had one advantage not shared 
by other oils, because mixtures of this sub- 
stance with Tween 80 and cell walls disperse 
instantly in saline to give stable emulsions. 

Preliminary tests were undertaken to de- 
termine whether the cell-wall vaccines con- 
tained any endotoxic factor which would be 
harmful if this product were used in man. 
Mice survived intraperitoneal inoculations with 
doses as high as 10 mg and 11-day-old-chick 
embryos were not killed by intravenously in- 
jected doses of 400 fig, the maximum dose em- 
ployed in contrast, 0.25 g of cell walls from 
several enteric Gram-negative bacteria con- 
stitutes as average median lethal dose for 
chick embryos. Likewise, 20 fig of Gram-nega- 
tive cell walls will evoke a typical endotoxic 



febrile response in rabbits whereas doses of 
mycobacterial cell walls as high as 1 mg did 
not produce significant fever. 

The potency of cell-wall vaccines as tested 
by the aerosol challenge method was shown 
to be dependent upon a specific cell-wall anti- 
gen which was shared in part by related 
species such as M. kansasii, M. avium, M. 
aquae, and M. butyricum but not by species 
such as Salmonella, Brucella, Listeria, and 
Corynebacterium pseudotuberculosis. The re- 
sults of these experiments also gave prelim- 
inary evidence that cell walls from H37Ra con- 
ferred a higher degree of immunity than any 
other preparations so far tested. 

Skin tests in rabbits and guinea pigs sensi- 
tized with living or killed whole cells or cell 
walls of M. tuberculosis, M. balnei, group I, 
II, III, or IV of the unclassified Mycobacteria 
were performed in continuing efforts to develop 
more specific antigens for use in detection 
and differentiation of mycobacterial infec- 
tions. As previously reported for M. tuber- 
culosis, M. phlei, and M. butyricum, proto- 
plasms and cell walls of these additional My- 
cobacteria give rise to delayed reactions when 
injected intradermally into sensitized rabbits 
or guinea pigs. The specificity of the reactions 
produced by cell walls and protoplasm differed. 
Minimal amounts of cell walls produced lesions 
in animals sensitized with either homologous 
or heterologous material. However, weaker dilu- 
tions of protoplasm which give rise to reac- 
tions in animals sensitized with homologous 
antigens usually would not elicit reactions in 
those sensitized with heterologous material. 

In collaboration with Dr. Leon Schmidt, 
safety tests of the standard cell-wall vaccinje 
enhanced with Drakeol have been initiated at 
the National Center for Primate Biology, Uni- 
versity of California at Davis, California. Be- 
cause of reactions at the site of inoculation, 
which were entirely unexpected, future po- 
tency tests of this product in monkeys have 
been delayed until further attempts are made 
to eliminate the factor responsible from the 
cell-wall vaccine. The safety test has been re- 
designed to provide preliminary information 
on the protective properties of this vaccine for 
monkeys. 



200 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Bordetella Pertussis Antigens 

Further studies on the histamine sensitizing 
factor (HSF) of B. pertussis were directed 
chiefly toward finding a practical method for 
isolating and purifying large quantities of this 
substance. Reasonably pure preparations have 
been obtained by magnesium sulfate precipi- 
tation of HSF from alkaline saline extracts of 
acetone-dried cells. HSF in Dupanol sensitized 
mice to histamine, to serotonin, to passively 
and actively induced anaphylaxis, and it in- 
creased antibody production by adjuvant ac- 
tion. This material was also found to induce 
hyperacute experimental allergic encephalomy- 
elitis (EAE) in rats that had received guinea 
pig cord suspensions. Thus, these preparations 
of reasonably pure HSF have most of the ac- 
tivity found in whole B. pertussis cells and 
possibly a single substance is responsible for 
most of these biological activities. 

B. pertussis strains are known to vary tre- 
mendously in their antigenic composition, as 
judged by agglutination and agglutination ab- 
sorption tests. Six specific antigens for B. per- 
tussis have been described. No significant dif- 
ferences in protective or histamine sensitizing 
ability were found among strains containing 
antigens 1, 3; 1, 2, 4; 1, 2, 3, 4; or 1, 2, 3, 4, 5. 
If immunity can be transferred passively with 
antigen-specific antiserums it would be possi- 
ble to identify the antigen associated with his- 
tamine sensitizing production and immunity 
in mice. 

Endotoxins 

The native hapten extracted from proto- 
plasm of Escherichia coli was studied further 
to determine whether this substance was 
indeed a precursor of endotoxin. This substance 
was found in nine of 11 strains of E. coli 
representative of four serotypes but none could 
be extracted from strains of Citrobacter freun- 
dii, Salmonella enteritidis, Salmonella typhi, 
and Serratia marascens. The molecular weights 
of native haptens isolated from these strains 
of E. coli averaged about 150,000; their physi- 
cal dimensions averaged about 15 A by 1200 A. 
In spite of the close serologic and chemical 
identity (sugar composition) of native hapten 



with endotoxin, it is unlikely that this ma- 
terial is a direct precursor of endotoxin. The 
physical dimensions could not conceivably be 
those of a subunit of endotoxin, and it does 
not behave like subunits derived from bile salt- 
dissociated endotoxin. 

The results of a study of the effect of sur- 
factants on endotoxins have provided addi- 
tional data in support of the theory of a micel- 
lar structure of endotoxin. When fully active 
endotoxin was treated with sodium desoxycho- 
late, subunits formed, with a molecular weight 
of 20,000 and dimensions of 10.5 A by 250 
A. The dissociated endotoxin was about 100- 
fold less potent in the pyrogenicity test in 
rabbits and it was no longer capable of stimu- 
lating an antibody response in rabbits. When 
the bile salts were removed by dialysis, the 
subunits reaggregated to form endotoxin par- 
ticles with a molecular weight of 500,000 and 
dimensions of 40 A and 1,000 A. The biologic 
activity of the dialyzed material was essen- 
tially restored. These results were consonant 
with the theory that the elements of endotoxin 
are composed of micellar aggregates of chain- 
like subunits. Also, it seems that the varying 
physical structures of isolated endotoxins re- 
flect only the end-to-end aggregation of basic 
elements produced by the extraction procedure 
or subsequent treatments. 

Microbial Proteins and Nucleic Acids 

Studies have been continued on the genetic 
relationships between various microorganisms 
as determined by the degree of polynucleotide 
similarity. Nucleotide base sequence homology 
between species was determined by mixing ra- 
dioisotope-labeled and sheared DNA fragments 
of one species with agar-embedded DNA of 
another species. 

Recently taxonomists have suggested that 
two new genera, Francisella and Yersinia, be 
added to the family Brucellaceae. Pasteurella 
tularensis and P. novicida would be placed in 
the genus Francisella and P. pestis and P. 
pseudotuberculosis in the genus Yersinia. P. 
tularensis and P. novicida were found to have 
a strong reciprocal cross relationship but to 
have no genetic relationship with P. pestis, P. 
pseudotuberculosis, or P. multocida. 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



201 



Biology of Microbial Agents in Arthropods 

This new project is concerned with the de- 
velopment of arthropod tissue cultures, their 
use for biologic studies of arthropod-trans- 
mitted pathogens, and with the development 
and behavior of pathogens in arthropod vec- 
tors. So far, attempts to develop a serially- 
propagating line of tick cells from metamor- 
phosing nymphal viscera have not been success- 
ful, but some explants have survived for 240 
days. Tick hemocytes cultures survived for an 
average of three weeks, with some retaining 
living cells for four months. Colorado tick fever 
virus was successfully grown in primary cul- 
tures of nymphal Dermacentor andersoni. By 
21-35 days after inoculation, the virus content 
of cell-free media had increased by as much 
as three to five logs, and in one instance virus 
was still detected for 126 days after inocula- 
tion. 

. Electron microscopic study of the subcellu- 
lar structure of Rickettsia rickettsi and R. 
prowazekii confirmed findings of previously 
reported studies that their internal structure 
closely parallels that of many bacteria. In in- 
fected ticks R. rickettsi were found in tissues 
of the hind gut, salivary glands, genital sys- 
tem and Malpighian tubules. Each organism 
was surrounded by a "halo" thought to be 
created by a tissue-digesting enzyme. 

LABORATORY OF BACTERIAL DISEASES 

Mycoplasma 

Mycoplasma organisms have been repeatedly 
isolated from the tissues of normal mice, par- 
ticularly from the brain and lung and in one 
instance, from blood. All of these isolates 
proved to be Mycoplasma neurolypticum. Sim- 
ilarly, M. neurolyticum also has been recov- 
ered from brain, lung, liver and enlarged 
lymph nodes of mice of several leukemia bear- 
ing strains, and from mice with transmissible 
leukemia atuopsied in the terminal stages of 
the disease. The possible role of M. neuroly- 
ticum and other mycoplasma in the origin of 
leukemia, and the role they may play in pro- 
ducing confusion and misinterpretation of ex- 



perimental results in leukemia research 
is being explored. 

Work on murine and other animal myco- 
plasma has led to the first identification of 
swine mycoplasma as tissue culture contam- 
inants. A mycoplasma isolated from murine 
leukemia cells in culture by a scientist at the 
Sloan-Kettering Institute was shown to be re- 
lated to strains of M. granularum, a serotype 
previously recovered only from swine. These 
studies also confirmed the work of other in- 
vestigators (LID) that some previously un- 
classified tissue culture contaminants were 
strains of M. hyorhinis, another serotype of 
swine origin. 

In a collaborative study with Dr. Leon 
Smith, St. Michaels Hospital, Newark, N.J., 
over 60 blood specimens from normal post- 
partum patients have been cultured. Only one 
recovery of mycoplasma was made in this 
series. This patient developed a low grade fever 
24 hours after delivery and M. hominis, type 
1, was recovered from the blood. There was a 
concomitant rise in antibody against M. hom- 
inis in the patient's serum. This finding pro- 
vides additional evidence of the pathogenicity 
of M. hominis, type 1, for humans, and indi- 
cates the necessity of a search for these 
organisms in fevers of unknown origin. 

In germfree guinea pigs inoculated with My- 
coplasma pneumoniae, the organisms were re- 
covered from the nasal passages with regu- 
larity over the six-week test period. Results 
with a high passage laboratory strain and 
with a recent human isolate were sim- 
ilar. There was no clinical disease nor gross 
pathological findings. Only low serum antibody 
titers developed. The germfree guinea pig does 
not appear to be a suitable host for the study 
of M. pneumoniae. 

In collaboration with the Perinatal Research 
Branch, NINDB, a study is under way to de- 
termine the normal mycoplasma flora of mon- 
keys. About 80% of the monkeys cultured have 
yielded mycoplasma. To date at least four types 
of mycoplasma have been isolated. Preliminary 
data indicate that at least one of these types 
may be similar to, or identical with, a type 
isolated from man. 



202 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Brucellosis 

As the program for eradication of bovine 
brucellosis has progressed, new and unexpected 
problems have arisen which threaten to im- 
pede progress toward total eradication of the 
disease. The question has arisen whether the 
use of Bmcella abortus, Strain 19 vaccine 
may be responsible for foci of infection in cer- 
tain problem herds, and whether the vaccine 
strain may actually be responsible for some 
infections. This Laboratory is collaborating in 



the study of unusual strains of Brucella 
isolated from wild and domestic sources in an 
attempt to clarify their ecological relation- 
ships. 

Studies on DNA homology in the genus 
Brucella were extended and completed. By the 
technique employed, DNA derived from smooth 
cultures of the three classical species, and from 
Br, neotomae, and rough Br. suis, cannot be 
differentiated one from another. The genus is 
thus highly homogenous and the S to R change 
was not reflected in the homology studies. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC 

DISEASES 



INTRODUCTION 

The following reports give some indication 
of the work of the NIAMD in the past year. 
Several features may be noted. There is a con- 
tinuing increase in interest and work in the 
field now known as Molecular Biology. This 
leitmotif of biological sciences in the 1960's 
has expanded to embrace work in clinical de- 
partments, pathology and biochemistry, and 
has also influenced the direction of work in 
the more physical and chemical sciences. In 
the latter case, the ready availability and the 
acknowledged biological importance of syn- 
thetic or purified polynucleotides and proteins 
(or their monomers) has stimulated quite 
basic studies of their physical and chemical 
properties. It seems reasonably certain that 
we are in the midst of a revolution in biology. 
Part and parcel of this revolution is the in- 
creasing interest in biological materials shown 
by chemists, physical chemists and theoretical 
chemists. And with this interest and activity, 
there has occurred an increasingly close col- 
laboration between physical and biological 
scientists. The answers to many fundamental 
problems in biology can only be found in the 
details of the chemical and physical interac- 
tions of the complex molecules found in living 
systems. It seems evident, therefore, that at 
NIH a systematic effort must be made to pro- 
vide facilities for a larger number of physical 
scientists, including those mainly concerned 
with theory. 

Engineering efforts to find ad hoc solutions 
to specific diseases cannot, it would appear, 
play more than a minor role in the eventual 
understanding of biological processes and con- 
trol of their disorders. The trend in the work 
of the scientists in NIAMD is clearly in the 



direction of investigation of biological ma- 
terials and processes at their most fundamen- 
tal level. 

It is not always apparent how many of these 
highly detailed and complicated studies of 
what seems to be very minor problems may 
have practical and clinical applications. Bac- 
teriophage, for example, may seem an unusual 
material for scientists to work upon, as these 
organisms only infect bacteria. Their extreme 
simplicity, however, (some strains have as few 
as a dozen genetic units compared to a hun- 
dred thousand or more in mammalian cells) 
permit accurate studies on the biochemical 
mechanisms of transfer of genetic information. 
This in turn serves as a model for virus in- 
fection of animal cells. Indeed bacteriophage 
infection of bacteria may well have significant 
implications in human disease as witnessed 
by the fact that diphtheria bacteria only pro- 
duce diphtheria toxin when they are infected 
with a certain bacteriophage. Hence, work in 
NIAMD in circular forms of DNA whose pres- 
ent function is unknown may well have impli- 
cations far beyond bacteriophage. 

On another level, the extensive studies on 
induction of enzyme synthesis in bacteria in 
NIAMD may appear unrelated to human dis- 
ease. However, unless we can understand this 
relatively simple biological system it seems 
clear that we will never understand how the 
human organism can adapt to the extraordi- 
narily varied diets people live upon, nor the 
dramatic situations in which certain cells 
change their pattern of adaptation and become 
cancerous. In fact, the best hope for eventual 
control of cancer in man is much more likely 
to derive from studies of the forces involved 
in the interaction of nucleotides in DNA than 
from empiric testing of anti-tumor drugs. 



203 



204 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Another major problem in human disease 
involves congenital abnormalities. There is 
some worry nowadays that, with improved 
methods of maintaining individuals possessing 
deleterious genes, we may eventually approach 
the situation in which the population is di- 
vided only into physicians and patients. A ra- 
tional approach to this problem seems to in- 
volve two factors: (1) differentiation, since 
errors in this process may lead to non-genetic 
congenital malformations and (2) mechanisms 
of disease production from deleterious genes. 
The first aspect is currently under study under 
defined conditions as the differentiation of the 
mammary gland. The hormones required, some 
of the biochemical reactions involved and their 
morphologic expressions have been elucidated. 
The exact role of the hormones and their mode 
of action, if they can be uncovered, could lead 
to practical approaches for preventing errors 
in differentiation — namely congenital anom- 
alies. 

The mechanism of the production of disease 
from genetic errors has already been partially 
clarified in many cases. In sickle cell anemia, 
not only the nature of the amino acid substi- 
tution which is the primary defect, but also a 
sensible chemical rationale for how this substi- 
tution produces the clinical picture of sickling 
have been advanced — largely by scientists now 
at NIAMD. The precise biochemical lesions 
in galactosemia, xanthinuria, homocystinuria, 
cystinuria, cystathioninuria, and gout have 
been elucidated over the past few years at 
NIAMD and elsewhere. In most cases a sensi- 
ble and effective therapy has been devised after 
the precise metabolic defect has been discov- 
ered. Just as importantly, tests have been de- 
vised for many of these inherited defects so 
that the healthy carrier can be identified. With 
this information there is hope that a positive 
eugenics type approach may eventually be 
able to reduce the incidence of these diseases. 

Many times apparently esoteric chemical pro- 
cedures are developed which appear to be of 
only theoretical significance. Recently, however, 
NIAMD scientists, by separating the optical 
isomers of certain morphine derivatives, were 
able to show that one of the isomers actually 



antagonized the dependency effects of mor- 
phine but retained analgesic activity. 

The fund of basic knowledge in the biological 
sciences is still immeasurably small compared 
to the rich and marvelously complex organiza- 
tion of structures and compounds that result in 
living cells. We can therefore anticipate that 
rational therapy for the serious diseases that 
beset mankind will await a clearer knowledge 
of biological processes. 

LABORATORY OF NUTRITION AND 
ENDOCRINOLOGY 

Studies on Folic Acid 

The catalytic and kinetic properties of the 
conversion of folic acid and dihydrofolic acid 
to the metabolically active tetrahydro-form 
have been investigated utilizing the highly 
purified dihydrofolic reductase from liver. At 
neutral pH values with TPNH as the reducing 
agent, the enzyme is completely specific for 
dihydrofolate, whereas folic acid and DPNH 
are inert as substrates. However, in the acid 
pH range, 4-5, both folic and dihydrofolic acid 
serve as substrates and DPNH may serve as 
the reducing agent. 

Kinetic analysis of the reaction indicated 
that the apparent affinity of dihydrofolic re- 
ductase for dihydrofolate is remarkably high. 
In addition, the interaction of dihydrofolate 
with the enzyme does not follow the expected 
"normal" kinetic behavior with respect to the 
velocity of the reaction vs. dihydrofolate con- 
centration. On the other hand, the interaction 
of folic acid with the enzyme exhibits "nor- 
mal" kinetics and the analysis suggests that 
folic acid is bound to the enzyme some 50 times 
less strongly than dihydrofolic acid. The inter- 
action of TPNH with the enzyme exhibits 
"normal" kinetic behavior at neutral pH when 
dihydrofolate is used as substrate. However, 
at acid pH values "abnormal" kinetics are 
noted using either dihydrofolate or folate. 
In addition, TPN was observed to be a potent 
inhibitor of the reductase reaction. Kinetic 
analysis demonstrated that the apparent affin- 
ity of the enzyme for TPN was almost the 
same as for TPNH. This is in line with the 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



205 



observation that TPN as well as TPNH is an 
effective stabilizing agent for the enzyme. 

Previously reported studies which described 
a marked activation of this enzyme by urea 
and organic mercurials suggested that dihy- 
drofolic reductase possesses many of the prop- 
erties of an "allosteric" enzyme. From this 
point of view, the "abnormal" behavior of the 
reaction with respect to dihydrofolate is not 
surprising since many of the "allosteric" en- 
zymes exhibit this type of "abnormal" kinetics. 
Thus the "normal" kinetic behavior observed 
when folic acid is used as the substrate correl- 
ates with the lack of significant stimulation 
of the enzyme by both urea and the organic 
mercurials in the presence of the fully oxi- 
dized folate, pteroylglutamic acid. 

The studies on the more complex and as yet 
unidentified forms of folic acid in tissues were 
directed to the stockpiling of a stable concen- 
trate and the development of chromatographic 
techniques to be used in further purification 
procedures to yield several components as pure 
compounds. (Drs. B. T. Kaufman and J. C. 
Keresztesy) 

Large-scale Processing of Biological Materials 

The large-scale laboratory continues to as- 
sist NIH investigators in procedures which re- 
quire processing and preparing materials in 
the volume too large for normal laboratory 
facilities. Fermentation operations were again 
this year in greatest demand. (D. L. Rogerson, 
Jr. and Dr. J. C. Keresztesy) 

Studies in Experimental Nutrition 

The studies on the development of liver ne- 
crosis in rats as influenced by various dietary 
components and state of "germfreeness" have 
been continued. It was found that many of the 
test animals may die without showing any, or 
very little, obvious damage to the liver cells. 
Other parameters for assessing liver damage 
are being explored. 

The possible role of the intestinal flora in 
the prevention of blacktongue in dogs was in- 
vestigated. The antibiotic, terramycin, was 
without effect. The administration of mixtures 
of antibiotics, together with the isolation tech- 



niques, are under study to influence intestinal 
flora in rats and dogs which will prove to be 
beneficial to the host. (Dr. F. S. Daft and 
E. G. McDaniel) 

Study of Protein Hormones 

Modifications of the structure of thyroid 
stimulating hormone (TSH), whether by en- 
zymic or chemical means destroy both biologi- 
cal and immunological activity. Physicochem- 
ical studies with bovine growth hormone have 
shown that in alkaline solution a monomeric 
form exists which again dimerizes upon acidi- 
fication. (Dr. P. G. Condliffe) 

Diabetes and Fat Metabolism 

The induction of diabetes in rats, by hor- 
mones from a transplantable pituitary tumor, 
was studied. Partial pancreatectomy was nec- 
essary to reduce the antidiabetic effect of en- 
dogenous insulin from the host's pancreas. 
Based on this finding partially pancreatecto- 
mized rats have been used to quantitatively 
study the hormonal interactions of ACTH and 
growth hormone upon the induction of dia- 
betes. A synergism was demonstrated. Immu- 
noassays of insulin in blood showed that the 
hormonally induced diabetes is not due to a 
fall in insulin level for actually the insulin 
level increased. (Dr. R. W. Bates) 

The metabolism of the diaphragm and fat 
tissue from rats after the induction of diabetes, 
by operative procedures, were investigated. 
The diaphragm muscle in vitro responded nor- 
mally to insulin but the adipose tissue was 
more insensitive to insulin. This latter partially 
accounts for the in vivo peripheral resistance 
to insulin found in some diabetics. (Dr. S. S. 
Chernick) 

Mechanism of Action on Isolated Fat Cells 

Using individual fat cells, it was demon- 
strated that many of the effects of hormones 
on the fat cells are at the level of the cell mem- 
brane. The hormones modify the permeability 
of the wall either directly or indirectly, e.g., 
lipolytic hormones release fatty acids which 
will alter cell permeability. The cell membrane 
may be altered sufficiently so that intracellu- 



206 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



lar enzymes are released to the medium. These 
effects are believed to be mediated by effects 
on the phospholipids in the cell wall. (Dr. M. 
Rodbell) 

Metabolism and Function of Fat Soluble 
Vitamins and Related Substances 

Lipid analyses of testes from eight species of 
animals including man showed characteristic 
fatty acid patterns, particularly in the polyun- 
saturated acids, for each species. These differ- 
ences could not be explained soley by the 
varying diets. Human testis had the highest 
content of the most unsaturated fatty acid (22 
carbons, 6 double bonds) found in tissues. 

A comparison of the lipid changes which oc- 
cur in rat testes damaged by deficiencies of 
vitamins A and E and also of zinc revealed 
that the most marked loss of phospholipid and 
alteration of the proportions of polyunsatu- 
rated fatty acids occurred in vitamin E defi- 
ciency. These results suggest a special involve- 
ment of vitamin E in lipid metabolism in the 
testis. 

The biopotencies and metabolism of the amine 
and N-methyl amine analogs of tocopherols we 
studied in chicks, a-tocopheramine was equally 
as active as standard «-tocopherol whereas (3- 
tocopheramine (like /3-tocopherol) was about 
one-third as active. N-methyl-/3-tocopheramine, 
however, was fully as active as /?-tocopherol as 
was also N-methyl-y-tocopheramine. The toco- 
pheramines were recovered unchanged from 
the blood and liver and were not converted to 
their tocopherols or quinones. (Dr. J. C. Bieri) 

Reversible Chemical Modifications of 
Cytochrome 

Salicylaldehyde was found to react specifi- 
cally with the lysine residues of cytochrome c 
to form a complex with reduced activity in the 
NADH reductase system and in the oxidation 
of ascorbate. Decrease of activity and also of 
solubility was proportional to the number of 
lysine groups involved (up to a total of 19 per 
molecule). Dialysis of the complex removed all 
salicylaldehyde and restored solubility and the 
ability to oxidize ascorbate but did not restore 
NADH-reductase activity until treatment with 



acid or alkali. The latter observation suggested 
the existence of polymers and chromatographic 
evidence for at least eight molecular species 
was obtained. (Dr. J. N. Williams) 

Relationship Between Purine-Pyrimidine 
Balance and Hepatic Fat Metabolism 

Perfusion studies comparing livers from nor- 
mal and orotic acid-fed rats have shown that 
the normal liver synthesizes and secretes into 
the perfusate ample quantities of fatty acids. 
The liver from the orotic acid-fed rat has a re- 
duced rate of fatty acid synthesis but more 
strikingly there is an almost complete block in 
lipid secretion into the perfusate. Further 
evidence that orotic acid interferes with lipo- 
protein formation was obtained by immuno- 
and paper electrophoretic analysis, ^-lipopro- 
teins were absent and a-lipoproteins signifi- 
cantly reduced in plasma from rats fed orotic 
acid. The disappearance of plasma /?-lipopro- 
teins was complete one week after administer- 
ing orotic acid but the effect was reversed by 
feeding adenine for only one or two days. (Dr. 
H. G. Windmueller) 

LABORATORY OF BIOCHEMISTRY AND 
METABOLISM 

Polysaccharide Metabolism 

Bacterial Systems 

Work on the 3-amino sugar isolated from 
Xanthomonas campestris has been completed. 
The crystalline preparation has been identified 
unequivocally as 3-acetamido-3,6-<dideoxy-D- 
galactose. Studies directed toward localization 
of this compound in the bacteria have been 
similarly completed with the demonstration 
that most, if not all, of the amino sugar is cova- 
lently bound to a second rare sugar, 6-deoxy- 
D-mannose, and is recoverable from the cell wall 
lipopolysaccharide. In addition, a sensitive and 
highly specific color reaction has been devel- 
oped which now permits the ready identification 
of N-acetylated-3-amino sugars in biological 
preparations. (Dr. G. Ash well) 

Current studies on the mechanism of bac- 
terial polysaccharide biosynthesis have resulted 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



207 



in the isolation of polysaccharide-deficient 
mutants. Work with these organisms has now 
provided the first preliminary evidence for the 
role of a lipid intermediate in exocellular poly- 
saccharide formation. (Drs. J. Hickman and 
G. G. Ashwell) 

Studies on the mechanism of deoxy sugar 
formation, utilizing- tritiated TDP-glucose la- 
beled specifically on carbon 3, have been com- 
pleted. This work has afforded new insight into 
the complex rearrangement accompanying 
deoxy sugar biosynthesis with the surprising 
demonstration that tritium from carbon 3 mi- 
grates uniquely and quantitatively to carbons 
2 and 6. Extension of this work is in progress 
whereby a new procedure is being developed for 
the chemical synthesis of tritiated glucose la- 
beled specifically in carbon 4. (Dr. 0. Gabriel) 

Animal Systems 

The conversion of glucose-6-P to inositol oc- 
curs in at least two steps. The first is a DPN- 
dependent cyclization of glucose-6-P to D-myo- 
inositol-1-P, and the second is a Mg + ""-depend- 
ent dephosphorylation to inositol. D-myo-inosi- 
tol-l-P, observed in nature for the first time 
during these studies, was isolated as a crystal- 
line compound after incubation of a dialyzed ex- 
tract of rat testis with glucose-6-P in the pres- 
ence of DPN, but in the absence of Mg ++ . 

The two steps in the formation of inositol 
are catalyzed by different enzymes, a cyclase 
and a phosphatase, separable on the basis of 
the fact that the cyclase is more heat labile 
than the phosphatase. Phosphatase free from 
cyclase has been prepared, but cyclase free 
from phosphatase has not yet been prepared. 
The phosphatase is highly specific for inositol 
monophosphates with equatorial conformation. 
Since the enzyme will not cleave glucose-6-P 
it can serve as an assay reagent for cyclase 
activity measured in terms of liberation of Pi 
from D-myo-inositol-1-P. 

Testis is 10-100 times as active as other tis- 
sues with respect to cyclase activity, while 
many tissues are at least as active as testis 
with respect to the phosphatase. (Dr. F. Eisen- 
berg, Jr., and Mr. A. H. Bolden) 



Galactogen, a polysaccharide present in the 
albumen gland of the snail, Helix pomatia, con- 
sists of D- and L-galactose units in a ratio of 
6-7:1. GDP-L-galactose has been isolated from 
an ethanol extract of these glands and is pre- 
sumed to be the precursor of L-galactose 
present in the polysaccharide. This nucleotide 
derivative of L-galactose is formed by the epi- 
merization of GDP-D-mannose. (Drs. E. F. 
Neufeld and E. M. Goudsmit) 

Glycoprotein Studies 

Antibodies 

The microsome-associated antibody that can 
be derived from the lymph nodes of immu- 
nized guinea pigs is being studied in order to 
determine the relationship of the microsome- 
antibody complex to antibody synthesis and se- 
cretion. Specifically, microsomes derived from 
guinea pigs immunized with dinitrophenyl- 
protein conjugates are complexed with anti- 
bodies, selective for the dinitrophenyl determi- 
nant, which can be specifically displaced by 
soluble normal gamma-globulins. Heavy chains 
derived from normal gamma-blobulins readily 
displaced microsomal-anti-dinitrophenyl anti- 
body from the microsome while the light 
chains were essentially inactive. Since all the 
covalently linked carbohydrate of gamma- 
globulin is known to reside in the heavy chains 
the role of the oligosaccharide moiety was ex- 
amined. Brief treatment of gamma-globulin 
with dilute sodium metaperiodate led to the 
isolation of a product that at y 5 the concentra- 
tion of untreated gamma-globulin was equally 
efficacious in the displacement assay. Thus far 
analysis has revealed that only the fucose and 
sialic acid are lost by periodate oxidation. A 
generalized nonspecific effect of periodate has 
been ruled out. 

When rabbit gamma-globulins are digested 
with pepsin at pH 4.5 at least five subfractions 
can be chromatographically resolved. An unre- 
solved digest of this kind was active in dis- 
sociating anti-dinitrophenyl-antibody from the 
microsome. The component (s) responsible is 
as yet unknown. 

The nature of the microsomal binding site 
of the anti-dinitrophenyl-antibody is to be 



208 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



analyzed by further studies of sub-fractions of 
normal gamma-globulins capable of displacing 
the specific antibody. (Drs. M. Kern and R. W. 
Swenson) 

Studies on Liver, Oviduct and Mastocytoma 
Glycoproteins 

Designed to study the mechanism by which 
sugar residues become attached to proteins. 
Many important secretory proteins contain co- 
valently bound sugars. Among them are a num- 
ber of hormones, most plasma proteins, blood 
group substances and epithelian secretions. The 
mode of glycosylation of these substances is of 
obvious importance. 

The transfer of N-acetylneuraminic acid- 
C 14 from its CMP derivative to an acceptor 
associated with liver microsomes was exam- 
ined. The transfer product was solubilized by 
sonication and subjected to Immunoelectro- 
phoresis. The endogenous microsomal acceptor 
was found to give precipitin lines with anti- 
serum prepared against rat serum, and is there- 
by tentatively identified as a mixture of pre- 
cursors of plasma glycoproteins. 

The transfer of xylose-C 14 from UDP- 
xylose-C 14 to endogenous acceptors was stud- 
ied in particulate preparations from oviduct of 
laying hens and in soluble enzyme preparations 
from mouse ascitic mast cell tumor P-815. 
Xylose was transferred to serine residues of an 
endogenous protein acceptor by one of the en- 
zyme fractions obtained from mastocytoma. 
This occurred in the absence of protein syn- 
thesis. Thus it is concluded that glycosylation 
of proteins begins after completion of the poly- 
peptide. While this concept has generally been 
accepted for the addition of sugars far re- 
moved from the polypeptide, this is the first 
demonstration involving the sugar adjacent to 
an amino acid residue. 

Besides catalyzing the formation of xylosyl- 
serine linkages, oviduct and mast cell tumor 
preparations transfer xylose to another, as yet 
unidentified, site, as evidenced by the forma- 
tion of alkali-stable, protein-bound xylose. 
(Drs. E. F. Neufeld, E. E. Grebner, P. J. 
O'Brien, and Mrs. C. W. Hall) 



Ribonucleic Acid 

Studies on RNase II 

The potassium-activated phosphodiesterase 
(RNase II) of E. coli was previously shown 
to be an exonuclease. The direction of hydroly- 
sis of polyribonucleotides by the enzyme has 
been investigated. Exonucleolytic attack can 
begin at that end of the molecule bearing an 
unesterified C-3'-hydroxyl group, or, alterna- 
tively, at that end bearing a C-5'-phosphomo- 
noester group. Furthermore, there are strong 
indications that the enzyme tends to hydrolyze 
a given chain more or less completely before 
going on to degrade another chain. That is to 
say, one chain is degraded at a time, rather 
than random hydrolysis of all the available 
chains. In order to study the possible role of 
RNase II in the degradation of messenger RNA, 
an in vitro protein synthesizing system that is 
free of detectable ribonucleases has been de- 
veloped. The system uses purified aminoacyl- 
transfer RNA, a soluble fraction from Lacto- 
bacillus arabinosus as a source of transfer en- 
zymes, and nuclease free E. coli ribosomes. In 
order to have ribosomes free of RNase I, a mu- 
tant devoid of that enzyme is used as a source 
of ribosomes. A technique to free these ribo- 
somes of adsorbed RNase II was developed. This 
method depends on the sedimentation of ribo- 
somes through a gradient of sucrose on a pre- 
parative scale. The ability of purified RNase 
II to degrade synthetic messenger RNA (i.e., 
poly U) when it is combined with ribosomes 
was then investigated. Little protection against 
degradation is afforded. In the course of these 
investigations a new method for the detection 
and preparation of poly U-ribosome complexes 
was discovered. The method depends on the 
fact that, under certain conditions, polyacryl- 
amide gels bind-free poly U, while poly U 
bound to ribosomes passes through the column 
with the ribosomes. (Drs. M. F. Singer, N. G. 
Nossal, N. M. Thanassi, and D. M. Logan) 

Studies on RNase I 

Release of RNase I from E. coli occurs to the 
extent of 50% as a result of osmotic shock. 
This probably represents the membrane-bound 
portion of the nuclease. The same procedure 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



209 



removes almost all of DNA endonuclease. Since 
these cells remain viable, they can be used for 
biological experiments. Encouraging results 
have been obtained on the infection of cells 
with isolated DNA. (Drs. N. G. Nossal, Y. An- 
raku, A. Weissbach, and L. A. Heppel) 

Thionucleotide Studies 

Biosynthesis of thionucleo tides (M. N. Lip- 
sett, A. Peterkofsky, and J. S. Norton) has 
been studied in a cell-free system. This pyri- 
doxal-requiring enzyme system will form 4- 
thiouridine from uridine residues in a pre- 
formed sRNA chain by transferring sulfur 
from a cystine donor. At least two other thio- 
nucleotides are also produced. This reaction 
represents a new type of structural modifica- 
tion introduced at the sRNA level, comparable 
to methylation and the pseudouridine rear- 
rangement. 

A pure species of E. coli tyrosine sRNA has 
been isolated, with the help of Dr. B. P. Doctor, 
and shown to contain two 4-thiouridine resi- 
dues per molecule. When this sRNA is con- 
verted to the disulfide state, the new disulfide 
bond has been shown to be intra- rather than 
inter-molecular. The amino acid accepting abil- 
ity of this sRNA is not affected by disulfide 
formation. (Dr. M. N. Lipsett) 

The sulfur moiety of 4-thiouridine in the 
sRNA molecule exhibits some but not all of the 
usual reactions of sulfhydryl groups. In the 
sRNA molecule, the hydrogen bonding of 4- 
thiouridine responds typically to denaturation 
with heat or urea. Mild oxidation of sRNA can 
form disulfide bonds between two such thionu- 
cleotides. These bonds are not denatured with 
heat or urea, but can be split with reducing 
agents. (Dr. M. N. Lipsett) 

Biochemical Studies of Lysogeny 

When the bacteriophage lambda, an induc- 
ible temperate phage, vegetatively multiplies 
in a host cell, it forms a new DNA exonuclease. 
This same type of nuclease has now been ob- 
served during the growth of the temperate 
phage 434 and this enzyme is being purified 
and studied. On the other hand, the virulent 
phage T4 does not produce the lambda type 



exonuclease. A study of other phages, such as 
noninducible temperate phage 299, the B. meg- 
aterium phages, etc., is under way to study 
their ability to produce new nucleases. 

The linear duplex DNA of phage lambda, 
when it enters a lysogenic cell, is rapidly con- 
verted to a circular, nonended form. In our 
work, mutants of lambda unable to lysogenize 
a host cell, were also found to form circles after 
superinfection of either sensitive or lysogenic 
cells. Studies on the requirements for the syn- 
theses of circular lambda DNA formed from 
superinfecting A, have shown that the circu- 
lar DNA can be formed from the linear form 
in the absence of any DNA synthesis. In addi- 
tion, lysogenic induction of E. coli K12A. leads 
to the formation of a circular DNA species, 
which is synthesized de novo after induction 
and appears to be a nonended lambda DNA 
molecule. This latter DNA species comprises 
only 1-2% of the total DNA being synthesized. 

Investigation of various mutants of phage 
lambda have shown that early mutants, blocked 
in the synthesis of lambda DNA, also cannot 
make circular DNA after lysogenic induction. 
Nevertheless, these mutants, when infecting a 
host cell, show a conversion of the linear 
lambda DNA molecule to a circular form. A 
survey of late mutants has shown that those 
which are capable of synthesizing linear 
lambda DNA after lysogenic induction will 
also make the circular DNA species. No mutant 
has yet been found which can only make cir- 
cular DNA or linear DNA exclusively. (Drs. 
A. Weissbach, W. E. Pricer, Jr., A. Lipton, 
and L. A. Salzman) 

Enzymes Near the Bacterial Surface 

Surface Enzymes and their Removal by Os- 
motic Shock 

A set of degradative enzymes is apparently 
located just under the cell wall and external to 
the protoplast membrane in E. coli. Evidence 
for this is: (1) histochemical studies (with 
Wetzel, Spicer) using the electron microscope; 
(2) the fact that enzyme activities can be 
measured with intact cells, although not com- 
pletely; (3) the selective release of these en- 
zymes by osmotic shock and spheroplast for- 



210 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mation while most of the proteins remain with- 
in the cell membrane. These surface enzymes 
occur not only in E. coli, but also in other 
gram-negative as well as in gram-positive 
bacteria. 

Several of the released enzymes have been 
extensively purified from the shock fluid. 
These include alkaline phosphatase and a spe- 
cific acid hexose phosphatase. Recently a new 
surface enzyme was discovered, which specifi- 
cally cleaves uridine diphosphoglucose (a 
UDPGase). Also, a specific protein inhibitor 
for 5'-nucleotidase and UDPGase has been 
found; its role in cell regulation is being in- 
vestigated. Both enzyme and inhibitor are be- 
ing purified. 

Osmotic shock involves exposing cells to 20% 
sucrose and ethylenediamine-tetraacetate 
(10 -3 to 10 M). Next, the mixture is cen- 
trifuged and the cell pellet is quickly dispersed 
in cold water or 5 x 10 " 4 M MgCl 2 . This pro- 
cedure causes cells to lose 4% of their proteins 
as well as nucleotides and amino acid pools, 
but they remain viable and recover after a lag 
period. A group of enzymes, including phos- 
phatases, RNase, DNase and UDPGase is se- 
lectively released. Shocked cells represent a 
useful biological tool. Experiments here and in 
other laboratories indicate temporary loss of 
ability to concentrate sugars. Thus, they can be 
used for study transport mechanisms and also 
the role of degradative enzymes. Further, the 
shocked cells have increased permeability to 
large molecules that ordinarily do not penetrate 
intact cells. 

Hexose phosphatase is one of the surface 
enzymes released by osmotic shock. Its level in 
the cell was widely varied and even completely 
suppressed by different growth conditions. The 
factors that control the concentration of this 
enzyme are being studied. (Drs. R. W. Brock- 
man, H. F. Dvorak, Y. Anraku, and L. A. 
Heppel) 

Enzymatic Utilization of Model Compounds in 
Bacterial Systems 

A group of enzymes involved in the utiliza- 
tion of L- and mesotartaric acid has been iso- 
lated and serves to outline the path of utiliza- 
tion of these compounds by strains of Pseudo- 



monas putida and Pseudomonas acidovorans. 
By a series of pyridine neucleotide-linked reac- 
tions, the tartaric acids are oxidized to dihy- 
droxyfumaric acid which, in turn, undergoes 
reductive decarboxylation to D-glycerate. Di- 
hydroxyfumarate, which is in equilibrium with 
its keto form, a /?-ketocarboxylic acid, is also 
readily decarboxylated spontaneously in aque- 
ous solution to yield a mixture of tartronic 
semialdehyde and hydroxypyruvic acid. The 
last two compounds are enzymatically reduced 
to form D-glyceric acid. The enzymes involved 
have been crystallized and are being studied 
from the viewpoint of mechanism of catalysis. 
(Drs. L. D. Kohn and W. B. Jakoby) 

The enzyme responsible for the formation of 
nicotinic acid mononucleotide (Drs. P. M. 
Packman and W. B. Jakoby) has been crystal- 
lized and has been shown to catalyze the fol- 
lowing reaction, the mechanism of which is 
under investigation: 



quinolinic acid + phosphorylpyrophosphat 

nicotinic mononucleotide + C0 2 + pyrophosphate. 

By extracting precipitates of highly purified 
proteins with solutions of varying degrees of 
concentration, a wide variety of proteins has 
been crystallized. Included are proteins with 
a molecular weight ranging from 30,000 to 
3,500,000, those with high and low polysac- 
charade content, proteins with a variety of 
catalytic function and even complexes of more 
than one protein. The method appears to be of 
general usefulness. One application of the 
technique (Dr. C. R. Steinman) is in the puri- 
fication of an aldehyde dehydrogenase, a type 
of enzyme enjoying ubiquitous distribution 
which is being brought to the stage where it 
may be studied on the protein level. 

Control of Phenotypic Expression of Genetic 
Information 

The Role of Galactose in the Induction of /3- 
galactosidase and Galactoside-permease in 
E. Coli 

In contrast to its reported role as a catabolite 
repressor, low levels of galactose have been 
found to stimulate the induction of /3-galacto- 
sidase in E. coli strains lacking galactokinase. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



211 



The stimulation does not occur in strains ca- 
pable of rapidly metabolizing galactose. This 
action of galactose is due to the induction of a 
hew permease for certain galactosides which 
is not induced by the usual inducers of the lac 
operon such as TMG,.IPTG and lactose. The 
assay for permease has been found to be ex- 
tremely sensitive to the osmolarity of the test 
solution; 10-20-fold increases in the sensitiv- 
ity of the determination have been achieved 
under conditions of relatively high hypotonic- 
ity. (Dr. I. G. Leder and Mr. J. W. Perry) 

Hormone-induced Differentiation of Mouse 
Mammary Gland in vitro 

Mammary gland explants from mice in the 
middle of their first pregnancy were organ- 
cultured on synthetic media to which insulin, 
hydrocortisone and prolactin were added. 
These hormones stimulate the synthesis of 
casein phosphoproteins after 48 hours of cul- 
ture to a rate several times greater than the 
initial rate of synthesis. Any of these hormones 
alone or in pairs elicited minimal or no stim- 
ulation. The effect is selective on the casein 
fraction of phosphoproteins. 

The synthesis of a-lactalbumin and /3-lacto- 
globulin is similarly dependent upon the pres- 
ence of all three hormones. On the other hand, 
the synthesis of soluble cytoplasmic nonmilk 
protein derived from mammary epithelial 
cells is stimulated maximally by insulin alone 
and is believed to reflect cell proliferation. 
There is a coordinate increase in the synthesis 
of electrophoretically separated casein compo- 
nents throughout mammary gland develop- 
ment. The increase in a-lactalbumin and /?- 
lactoglobulin synthesis is not coordinate 
throughout mammary gland development; as 
the lactational state is approached the a/p 
ratio increases. In vitro the 3-hormone combi- 
nation effects stimulation of casein and whey 
protein synthesis of the same magnitude, with 
the same time course, and the ratio of whey 
proteins to casein synthesized corresponds 
well with the ratio found in mouse milk. 

It has been concluded that a pool unphos- 
phorylated or incompletely phosphorylated ca- 
sein exists in mammary gland, and that phos- 



phorylation of the polypeptide chains is a ma- 
jor mechanism for incorporation of phosphate 
into casein. Human placental lactogen has been 
found to have the same activity as prolactin 
in this system. 

The selective biosynthetic responses to the 
hormones are parelleled by specific histologi- 
cal changes in the alveolar epithelium of the 
gland. Thus tissue cultured in the three hor- 
mone system is distinguished from tissue cul- 
tured in systems lacking one or more hormones 
by the characteristic orderly arrangement of 
epithelial cells of each alveolus, the very large 
nucleoli, and the accumulation of a great deal 
of stainable material in the alveolar lumina. 
The time course of all these histological and 
cytological changes proceeds in parallel with 
the synthesis of milk proteins. 

Evidence has been obtained which indicates 
that the hormone-dependent induction of the 
synthesis of milk proteins requires the prior 
synthesis of DNA by the epithelial cells. (Drs. 
Y. J. Topper, F. E. Stockdale, R. W. Turking- 
ton, and D. H. Lockwood) 

Reduction of Protein Disulfide Bonds by Liver 
Enzyme Systems 

Mammalian liver is known to possess en- 
zyme systems which catalyze the reduction of 
disulfide bonds in single peptides and proteins. 
The enzymes have been conventionally classi- 
fied either as transhydrogenases (e.g., gluta- 
thione-insulin transhydrogenase, GIT) for 
which a small thiol may serve as primary re- 
ductant, or as reductases (e.g., glutathione re- 
ductase) for which some other small molecule 
(e.g., TPNH) can serve as hyrdogen donor. 
Studies of the action of GIT on insulin have 
been continued. Spectrophotometric evidence 
indicates that GIT can itself be reduced by 
GSH. The fact that such thiol-treated enzyme 
is strongly inhibited by sulfhydryl reagents, 
in contrast to untreated enzyme, supports the 
idea that the groups thus reduced are disulfide 
bonds. These observations have further sug- 
gested that a reduced, thio-containing form of 
the enzyme may serve as active intermediate 
in the action of this enzyme on insulin and 
other proteins. (Dr. F. Tietze) 



212 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



LABORATORY OF CHEMISTRY 

Section on Biochemicals Mechanisms 

Electrolytic Cleavage of Tyrosyl-Peptide Bonds 
and the Mapping of Tertiary Structure 

A novel technique has been developed for 
conducting electrolytic oxidation or reduction 
on sensitive or polyfunctional substances. A 
buffered solution of an enzyme is caused to 
flow continuously in a thin film over a rapidly 
rotating cathode or anode. By regulation of 
voltage and rate of flow, the degree of oxida- 
tion or reduction can be controlled at will. Thus, 
with ribonuclease at pH 5, cleavage of the pep- 
tide bonds following the three exposed tyro- 
sines is effected specifically, while the three 
buried tyrosines remain intact. Similar stud- 
ies on disulfide reduction are in progress. By 
combining the data from a series of such ex- 
periments, it should be possible to achieve 
partial mapping of the surface of an enzyme 
in solution. (L. A. Cohen, L. Farber, and S. 
Isoe) 

Synthetic Models for Hydrolytic Enzymes 

Whether amides acylate on nitrogen or oxy- 
gen is usually difficult to ascertain by spectro- 
scopic methods. In the case of the tautomeric 
system of cycloserine, it has been virtually im- 
possible to reach a decision; however, the 
problem has now been solved by means of nu- 
clear magnetic resonance spectroscopy. The in- 
troduction of a double bond into the five-mem- 
bered ring alters dihedral angles of the ring 
substituents to a degree sufficient to allow a 
distinction to be made. Thus, acetylation leads 
to the N-acetyl derivative, whereas tosylation 
occurs on oxygen. Such information is needed 
to help in assessing the role, if any, of the pow- 
erful nucleophile, hydroxylamine, in hydrolytic 
enzyme mechanisms. (L. A. Cohen, G. W. 
Milne, and C. R. Gunter) 

Oxidation Mechanisms in Metabolic Processes 

The catalytic roles of ubiquinone and Vita- 
min K in oxidative phosphorylation require 
that a pathway be available for the direct re- 
ductive cyclization of quinone to chromanols 
under mild conditions. It has been possible to 



demonstrate, in vitro, such a direct reduction 
by means of sulfhydryl groups. Dihydrolipoic 
acid was found to be a particularly effective 
reducing agent. Of special interest is the fact 
that this reaction is favored by non-polar 
media, as would exist in the lipid particles of 
mitochondria, the site of oxidative phosphory- 
lation. (L. A. Cohen and M. A. Oxman) 

Section on Carbohydrates 

Studies Bearing on the Presumed Ester Link- 
age in Certain Glycoproteins 

The lability toward alkali of the N° acetyl- 
galactosamine residues in ovine submaxillary 
gland mucoprotein has led to the suggestion 
that this sugar (and the sialic acid residue at- 
tached thereto) is attached to the nonpepti de- 
bonded carboxyl groups of glutamic and as- 
partic acids by an ester linkage involving C-l 
of the amino sugar. Since no authentic 2-acet- 
amido-l-O-cyl-2-deoxyhexoses were known, 
it became of interest to synthesize representa- 
tives of this class of substances and to examine 
their properties. Using techniques developed 
earlier by Dr. R. Harrison, Dr. T. D. Inch was 
v able to prepare a variety of 2-acetamido-l-O- 
acyl-2-deoxyhexopyranose derivatives of the D- 
glucose and D-galactose series. The behavior 
of six 2-acetamido-l-0-acyl-3,4,6-tri-0-benzyl- 
2-deoxyhexopyranoses with alkali was studied. 
Under conditions which cleave the carbohy- 
drate residues from ovine submaxillary gland 
mucoprotein at a measurable rate, these syn- 
thetic substances hydrolyze much too fast for 
measurement. Indeed, the substances autalyzed 
at a rate convenient for measurement at 50° in 
aqueous dioxane and in the absence of alkali. 
These results may be viewed as casting some 
doubt on the presence of ester linkages in gly- 
coproteins. 

Not only do the 2-acetamido-l-0-acyl-2-de- 
oxyhexopyranoses readily hydrolyze in the ab- 
sence of added catalyst but they also react with 
alcohols. Those having an acyloxy function at 
C-l cis to the acetamido group at C-2 undergo 
simple transesterification to give the free 2- 
acetamido-2-deoxyhexose while those with a 
trans arrangement give a trans glycoside. This 
simple, stereospecific and remarkable reaction 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



213 



has not been observed before; it is not shown 
by the fully acylated 2-acetamido-2-deoxyhexo- 
pyranoses. In a study of the solvolysis of 2,3,5- 
tri-O-benzyl-a-D-arabinofuranosyl chloride, 2, 
3-di-0-benzyle-5-0-p nitrobenzoyl-«-D-arabino- 
furanosyl chloride, and 2-0-benzyl-3,5-di-0-?? 
nitrobenzoyl-a-D-arabinofuranosyl chloride, 
Dr. C. P. J. Glaudemans has shown that acyl 
groups far removed from C-l may inhibit the 
solvolytic removal of groups from C-l. It is 
possible that the failure of fully acylated 2- 
acetamido-2-deoxyhexopyranoses to give glyco- 
sides when treated with alcohols may be at- 
tributed to the deactivating influence of the 
acyl groups at C-3, C-4, and C-6. 

The readiness with which trans-2-acetamido- 
l-O-acyl-2-deoxyhexopyranoses react with al- 
cohols suggests that analogous esters (of glu- 
tamic and aspartic acid) may serve as inter- 
mediates in the formation of the O-glycosides 
(of serine and threonine) which have been 
shown to be present in some gylcoproteins. 

N,N-Diacylhexosamines and the Conformation 
of Amino Sugars 

In the course of the above research (a new 
type of amino sugar derivative) the 2-diacyl- 
amino-2-deoxyhexopyranoses was discovered 
by Dr. Inch. An extensive investigation of the 
chemical behavior of 2V,2V,-diacyl derivatives of 
the hexosamines was undertaken. Particular 
attention was devoted to 2-(A7"-acetylbenzam- 
ido)-2-deoxy-D-glucose. On heating in chloro- 
form solution, this substance undergoes N — 
acyl migration and gives at least three prod- 
ucts: l-0-acetyl-2-benzamido-2-deoxy-«-D- 
gluco-pyranose, 2-acetamido-l-0-benzoyl-2-de- 
oxy-a-D-glycopyranose, and 2-acetamido-l-O- 
benzoyl-2-deoxy-a-D-glucofuranose. Benzoyl 
migration predominates over acetyl migration 
in this case. 

The conformations of the ordinary acety- 
lated forms of the common N-acelylhexosa- 
mines (the 2-acetamido-l,3,4,6-tetra-0-acetyl- 
2-deoxyhexopyranoses of the D-glucose, D-ga- 
lactose, and D-mannose series) are not 
apparent from the nuclear magnetic resonance 
spectra of these substances. In contrast, the 
n.m.r. spectra of the corresponding substances 
with two acyl groups attached to nitro- 



gen are readily susceptible to conformational 
analysis. Drs. Inch and J. R. Plimmer have 
thereby 2-acetamido-2-deoxy-D-mannopyranose 
(iV-acetyl-D-mannosamine) derivatives most 
probably do not exist in the C-l conformation 
which is normal for 2-acetamido-2-deoxy-D- 
glucopyranose and D-galactopyranose. 

Studies Toward the Synthesis of Polyvinyl 
Glycosides 

In view of the fact that the serological prop- 
erties of many natural substances depend upon 
the nature of the external carbohydrate resi- 
dues rather than on the backbone structure to 
which they are attached, attention has been 
turned to the synthesis of glycosides of poly- 
vinyl alcohol. Since glycosylation of polyvinyl 
alcohol is beset with numerous and apparently 
insuperable difficulties, the study of the syn- 
thesis of monomeric materials, the two vinyl 
D-glucopyranosides was undertaken. This work 
was initiated by Mr. T. D. Perrine in the 
Rocky Mountain Laboratory, Hamilton, Mon- 
tana. Starting with the known 2-chlorethyl 2,- 
3,4,6-tetra-0-acetyl-j8-D-glucopyranoside, Mr. 
Perrine succeeded in synthesizing (by a con- 
siderable number of steps) the hitherto un- 
known vinyl 2,3,4, 6-tetra-0-acetyl-/?-D-gluco- 
pyranoside. Using a wholly different approach, 
Dr. C. P. J. Glaudemans was able to convert 
2,3,4,6-tetra-O-benzyl-D-glucopyranose into the 
two anomeric vinyl 2,3,4, 6-tetra-O-benzyl-D- 
glucopyranosides and showed that these sub- 
stances could be polymerized through the ac- 
tion of boron trifluoride. Dr. R. K. Ness was 
able to dibenzylate these two substituted gly- 
cosides and thus obtain, for the first time, the 
anomeric vinyl D-glucopyranosides. It is of 
some theoretical interest to note that, while 
the a anomer is comparatively stable to the 
action of alkali, the /? anomer is converted into 
l,6-anhydro-/3-D-glucopyranose; thus the be- 
havior of the vinyl D-glucopyranosides paral- 
lels that of the phenyl D-glucopyranosides. 

Higher-Carbon Sugars 

The second nonulose from the avocado, which 
had been identified earlier as Ti-erythro-lj- 
galacto-nonulose through degradation experi- 



214 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ments, has now had its identity confirmed by 
two methods of synthesis. Both the natural 
and synthetic nonuloses were reduced to the 
same pair of nonitols and the structures of 
these were proved through oxidation with 
Acetobacter suboxydans. (H. H. Sephton) 

Studies on the higher-carbon sugars and 
other carbohydrates in Sedum species and 
Pichi tops (Fabriana imbricata) were contin- 
ued. (N. K. Richtmyer and E. W. Tracy) 

Thio Sugars 

D-Glucose diphenyl dithioacetal was pre- 
pared for the first time, and converted into 
phenyl 1-thio-a-D-glucopyranoside and phenyl 
1-thio-a-D-glucofuranoside; the glucosides were 
then converted into the corresponding sulfones, 
and an «-D-glycofuranosyl phenyl sulfoxide 
was obtained, thought only in very small yield. 
(E. Zissis) 

Amino Sugars 

Several 1-amino-l-deoxyheptitols were pre- 
pared and preliminary studies showed that 
some of them, at least, could be oxidized by 
Acetobacter suboxydans to produce 7-amino-7- 
deoxyheptuloses. (H. J . F. Angus) 

Section on Medicinal Chemistry 

Evaluation of Analgesics 

Dose-range finding experiments were per- 
formed on 43 drugs most of which were from 
external sources. Of these, 42 were subjected 
to complete analgesic assay (Eddy modifica- 
tion of Woolfe-Macdonald hot-plate method) 
and acute toxicities were determined for 10. 
Nearly half of the 42 drugs assessed were sent 
to the University of Michigan for physical de- 
pendence studies in rhesus monkeys. Note- 
worthy results are the discovery of antagonism 
to morphine-like effects for two Zevo-isomers 
of the benzomorphan series which are strong 
analgesics in their own right, and of codeine- 
like physical dependence capacity and analge- 
sic activity for the corresponding dextro- 
isomers usually inert in both respects. As each 
racemate had no physical dependence capacity 



(indicated in the 1964-1965 report), the levo- 
isomers must nullify the physical dependence 
property of their dextro counterparts as well 
as some of the pharmacologic effects of mor- 
phine. (E. L. May, N. B. Eddy, L. Atwell and 
W. Ness) 

In Vitro Screening Methods for Anti-Inflam- 
matory Properties 

Based on the assumption that there is ab- 
normal release of histamine through decar- 
boxylation of histidine in the inflammatory 
process a commercial bacterial enzyme from 
CI. welchii was found to decarboxylate histi- 
dine at 37°. However, aspirin, phenylbutazone 
and hydrocortisone had no inhibitory effect on 
this decarboxylation. 

Albumin Coagulation. Mizushima's method, 
the suppression of heat-coagulation of certain 
globulin fractions by the standard anti-inflam- 
matory agents, was found to be unreliable inas- 
much as the solvent-buffer system which had 
to be used for phenylbutazone, indomethacin 
and hydrocortisone hemisuccinate gave almost 
as much suppression as the known anti-inflam- 
matory agents. 

Albumin-Trinitrobenzaldehyde Complex. The 
method of Whitehouse, the displacement of 
2,4,6-trinitrobenzaldehyde from albumin (pre- 
sumably from the terminal amino groups 
of lysine) by presently used anti-inflammatory 
agents results in a change in absorption at 425 
and 526 m^. Phenylbutazone and indometha- 
cin were indeed positive but a definite draw- 
back of the method is the quick deterioration 
of the trinitrobenzaldehyde in the buffer used. 
There was also too rapid change in transmis- 
sion at the wavelengths used for quantitative 
estimations. This was considered to be the best 
of the three methods tried. (A. E. Jacobson) 

Optical Resolutions of Benzomorphans 

Because of the finding that «( — )-5,9-diethyl- 
2'-hydroxy-2-methyl-6,7-benzomorphan and /?- 
( — )-2,9-dimethyl-2'-hydroxy-5-phenyl-6,7-ben- 
zomorphan are antagonists for their dextro 
counterparts and certain effects of morphine, 
a-2, 9-dimethyl-2'-hydroxy-5-propyl-and 5-ethyl- 
ing resolved by various optically active acids. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



215 



2'hydroxy-2-methyl-6,7-benzomorphans are be- 
Mandelic acid appears to be superior to ( + )-10- 
camphorsulfonic acid and ( + ) -3-bromocam- 
phor-8-sulfonic acid. (E. L. May, A. E. Jacob- 
son, and J. H. Ager) 

Abnormal Stevens Rearrangement 

Rearrangement of l-p-chlorobenzyl-trimethyl- 
1,2,5, 6-tetrahydropyridinium chloride (PhLi) 
has given in addition to two expected prod- 
ucts (2- and 4-p-chlorobenzyl-l,3,4-trimethyl- 
1,2,5,6-tetrahydropyridines) , 2-p-chlorophenyl- 
l,3,3-trimethyl-4-methylenepiperidine. This 
third product is believed to be formed by ab- 
straction of a benzylic proton followed by pyri- 
dine ring cleavage of the original quaternary 
to give an allylic carbanionpyridinium dipolar 
ion. Proton transfers and rotations about single 
bonds can lead to a different allylic carbanion 
which then ring closes. A comparable product 
is obtained when the p-position is unsubstituted. 
(A. E. Jacobson) 

Rearrangement Products 9-Acetoxy- and -hy- 
droxy '-benzomorphans 

Pyrolysis of 9-acetoxy- and 9-hydroxy-2,5,9- 
trimethyl-6,7-benzomorphan perchlorate at 
185° gives a mixture of l,2,3,4-tetrahydro-l,4, 
4-trimethyl-9H-indeno [2,1-b] pyridine (II), 
the yields and proportion depending upon py- 
rolysis time with short periods favoring II. As 
II (after isolation) could not be converted to 
I, a phenonium-ion intermediate from which 
II could be formed irreversibly and I reversi- 
bly, is implicated. Thionyl chloride treatment 
of 9-hydroxy-2,5-9-trimethyl-6,7-benzomorphan 
gave a 4% yield of II. (R. T. Parfitt, E. M. 
Fry and E. L. May) 

A New Aromatization Method for fi-Tetralones 
and a New Route to 2-Aminonaphthalenes 

The pyrrolidine enamine of /3-tetralone re- 
acts with CNC1 to give l-cyano-/?-tetralone. 
The same enamine with CNBr yields l-(2- 
naphthyl-pyrrolidine). This has been extended 
to the preparation of other tertiary and sec- 
ondary 2-aminonaphthalenes. The yields are 
uniformly good. (R. T. Parfitt) 



Schiff -Bases and Oxazolidines 

Condensation of N-methyl-4-piperidone (see 
1964-1965 report) with 2-aminoethanols (pri- 
mary and secondary) leads to reasonably 
stable spiro-oxazolidines with only a small 
percentage of tautomeric Schiff-base (in the 
case of pirmary amines) in equilibrium. With 
aromatic aldehydes only the Schiff bases exist. 
Both the Schiff bases and oxazolidines can be 
hydrogenated to substituted /?-amino alcohols 
which are of interest in malaria and cancer 
chemotherapy. (J. H. Ager) 

Potential Aldolase Inhibitors 

Osmium tetroxide catalyzed hydroxylations 
of 1,4-dimethylenecyclohexanes were com- 
pleted. Three diols resulting from this study 
are undergoing testing for antiviral activ- 
ity. The preparation and characterization 
of 2-hydroxy-3-oxobutylphosphate have been 
achieved, and evidence for the isomeric 3- 
hydroxy-2-oxobutyl phosphate is at hand. This 
study is expected to point the way to increas- 
ing the accessibility of the biologically sig- 
nificant substances, dihydroxyacetone phos- 
phate and glyceraldehyde phosphate hitherto 
accessible only as their dimethyl ketals. (J. G. 
Murphy) 

Immunologic Studies in Clinical Hematology 

Quinidine was catalytically reduced to dihy- 
droquinidine and the latter O-demethylated to 
dihydrocupreidine. This crude phenolic base 
was then converted to 6 aminodihydrocin- 
chonine via the Bucherer reaction. Diazotiza- 
tion and compling with normal human serum 
gave a diazoprotein which has been tested for 
antigenic activity. (L. J. Sargent) 

Dihydrocodeinone Isomers 

The report of the formation of a "second" 
isomer during cyclization of 1,7-dibromodihy- 
drocodeinonedihydromethine has been found 
to be in error. The "second" substance, recov- 
ered in less than 5% yield, is now known to be 
starting bromo ketone while the main product 
appears to be the 7-membered heterocycle re- 



216 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



suiting from nitrogen closure to the 7-position 
rather than to the 5-position. (L. J. Sargent) 

Polyethylenimine Studies 

Cyanoethylation of polyethylenimine (PEI) 
in pyridine gives a quantitative yield of tract- 
able material with the expected empirical for- 
mula. However, spectroscopic and other evi- 
dence indicate that this material is in part 
altered at the cyano groups which can be hy- 
drolyzed to only 50% of the expected amide 
groups. Copper-binding studies give definite 
indication that PEI has a microgel-type struc- 
ture. Its behavior is entirely different from 
that of the isomeric polyvinylamine which is 
probably linear. Moreover, added chloride ion 
gives spectral changes which, although slight, 
offer promise of use in determining the ratio 
of primary to secondary to tertiary anime 
groups in the polymer, in turn a measure of 
its branching. (T. D. Perrine) 

Synthesis of i-p-methoxy-l-vinyl-2-pyrrolidone 

The material described in the 1964-1965 re- 
port as 4-p-methoxyphenyl-l-(2-dimethyla- 
minoethyl)-2-pyrrolidone (I) was found to be 
a mixture of three products by thin-layer 
chromatography. Column chromatography ef- 
fected separation of these products one of 
which was identified as I through the methio- 
dide which was subjected to Hofmann elimi- 
nation with silver oxide to give 4-p-methoxy- 
phenyl-l-vinyl-2-pyrrolidone for polymeriza- 
tion. ( W. R. Landis and T. D. Perrine) 

Naphth- and Benz-3 ,4-dihydro-oxazines 

Reaction of salicylic acid with dicylohexyl- 
carbodiimide in a variety of nonpolar or 
slightly polar solvents gives a 20% yield 
of 3-cyclohexyl-2-cyclohexylimino-3,4-dihydro-4- 
0x0-2H-l,3-benzoxazine parallel with an 
earlier finding with l-hydroxy-2-naphthoic 
acid which yielded the corresponding naphth 
[2,l-e]-l,3-oxazine. Lithium aluminum hydride 
reduction of the naphthoxazine gives 3-cyclo- 
hexyl-3, 4-dihydro-2H-naphth [2,l-e]-l,3-oxa- 
zine which is superior to aspirin, salicylamide 
and the 3-phenylsalicylamides as an analgesic 
(mouse-hot plate assay method) and whose 



structure was confirmed by synthesis. (E. L. 
May) 

Section on Microanalytical Services and 

Instrumentation 

Approximately 11,000 chemical and instru- 
mental analyses were performed for about 150 
research scientists of NIH and for several sci- 
entists in other government agencies, includ- 
ing Nava 1 Medical Research Institute, Food 
and Drug Administration, and Department of 
Agriculture. These analyses, performed by 8 
chemists and technicians, included many non- 
routine determinations which required more 
than ordinary time and effort. Special 
assistance was given to many research workers 
in relation to work carried out in their own 
laboratories. The acquisition of a Perkin- 
Elmer Model 421 Infrared Spectrophotometer 
has improved the quality of service rendered 
in this area. 

Section on Steroids 

Study of Alkaloids from Solanum congesti- 
florum (natri) 

The isolation of (25R)-22,26-imino-5a-cho- 
lest-22(N)-en-3/?-ol (I) as the major steroidal 
alkaloid present in S. congestiflorum has led 
us to the study of other alkaloids present in 
the plant. The two others so far identified have 
proven to be the 16«-hydroxy-(II) and the 
24-oxo (III) derivative of I. It has been found 
that I and II in the plant exists as glycosides 
with the sugars glucose, galactose and rham- 
nose. The isolation of the alkamine I is of con- 
siderable interest biogenetically since most of 
the Solanum and Veratrum alkaloids could con- 
ceivably arise from I by simple ring closure 
of hydroxylation and ring closure etc. Bioge- 
netic experiments along these lines are con- 
templated. (Y. Sato, Y. Sato and R. Overton) 

Synthesis and Reactions of Heteroisteroids 

Novel heterocyclic steroids possessing bio- 
logical activity are being actively sought. It 
was found that the lithium aluminum hydride 
reduction of 3'-methyl-isoxazolino [17,16d] 
-5-androsten-3/?-ol ('65 report) affords the 16- 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



217 



hydroxy-20-amino steroid in good yields. This 
constitutes an expedient procedure for the 
preparation of this intermediate. Fairly pure 
thiosemicarbazones of estrone, estrone methyl 
ether and their N-acetyl derivatives, which is 
said to offer some difficulty in preparation, 
was prepared for the Endocrinology Branch, 
NCI for biochemical studies. Related steroid 
thiosemicarbazones were prepared for antiviral 
activity. (Y. Sato and R. Overton) 

Mass Spectrometry of Steroidal Glycosides 

The trimethylsilyl ether derivative of sola- 
sodine monoglucoside prepared with SIL- 
PREP/dmf reagent gives rise to molecular 
ion peaks corresponding to the tetra and penta- 
(trimethylsilyl) ether derivatives in the mass 
spectrum. Studies are continuing to determine 
the feasibility of utilizing the method for de- 
termination of molecular weights of steroidal 
glycosides. (Y. Sato, Y. Sato and H. Fales) 

Sterols from Parasites 

Exploratory data indicate that the parasites 
Trypanosoma cruzi grown on blood free media 
contain cholesterol and/or ergosterol. No 
sterols were found in the media. (R. Overton, 
Y. Sato and T. von Brand) 

Photor -eduction of /3-Estradiol 

Irradiation of a solution of /3-estradiol and 
sodium borohydride in ethanol with a high 
pressure mercury lamp (Vycor filter) gave 
3a,17/3-dihydroxy-5(10)-estrene as the major 
monomeric product. Two other monomers and 
polymeric material were also obtained. (J. A. 
Waters, M. Chaykovsky and B. Witkop) 

Chemistry of Iresine celosia 

Twelve compounds were isolated from the 
crude plant material. Preliminary chemical in- 
vestigations were made and several fractions 
containing these compounds were submitted for 
biological evaluation (epilepsy and cancer 
chemotherapy). South American medical au- 
thorities have reported the value of the crude 
plant in treatment of certain types of these 
diseases. (J. A. Waters and Y. Sato) 



Sterol Biosynthesis in Plants 



Radioactive stigmasterol and /3-sitosterol, 
isolated from soya bean plants following incu- 
bation with mevalonic acid-2-C 14 , were rein- 
cubated with excised soya bean leaves. Exten- 
sive analysis by thin-layer chromatography 
and scanning for radioactivity indicated that 
stignasterol and /^-sitosterol are not directly 
related biosynthetically via hydrogenase-dehy- 
drogenase enzyme systems. (D. Johnson and 
J. A. Waters) 

Metabolism of C u -labeled Steroids by Adren- 
als from Rats with a Mammotropic Pi- 
tuitary Tumor 

Radioactive progesterone, corticosterone, 
and deoxycorticosterone respectively were in- 
cubated in vitro with adrenal slices from 
normal rats and rats subjected to long-term 
stress of tropic hormones of the anterior pi- 
tuitary. Comparisons of the radioactive met- 
abolite patterns, as measured by column chro- 
matography and thin-layer techniques, has 
revealed significant abnormalities in steroid 
biogenesis by the stressed rat adrenal. The 
results obtained suggest that enzymatic de- 
ficiencies or stimulation in the adrenal may 
have resulted from the prolonged effects of 
ACTH, growth hormone, and prolactin. (D. 
Johnson and D. Francois) 

Adrenal Metabolism of the Gerbil 

Progesterone-4-C 14 was incubated with ger- 
bil adrenal tissue in vitro and the radioactive 
metabolites were analyzed by column chroma- 
tography and thin-layer techniques. Chroma- 
tographic identity with known compounds 
revealed the presence of 11-deoxy corticoster- 
one, 18-hydroxy-ll-deoxycorticosterone, and 
cortisone as progesterone metabolites. A dif- 
ference in metabolite pattern was observed, 
depending on the use of ether or Nembutal as 
anesthetic. These studies indicate that the ger- 
bil is well suited to adrenal steroid investiga- 
tions and that its adrenal metabolism is 
different from that of the rat. (D. Francois 
and D. Johnson) 



218 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Studies on the Biosynthesis and Metabolism 
of Biogenic Amines 

The neuro-transmittor, norepinephrine, 
arises from the amino acid, tyrosine, through 
the sequential action of three enzymes. The 
biosynthesis of norepinephrine plays an im- 
portant role in the functioning of the sympa- 
thetic and central nervous system. The localiza- 
tion both intracellularly and grossly of this 
biosynthetic trio of enzymes is under investi- 
gation. (C. R. Creveling, B. Witkop and J. 
Daly) 

Section on Metabolites 

Batrachotoxin, the Strongest Venom Known 

It has now been found that the major toxic 
principle of the Colombian arrow poison frog 
(Phyllobates latinasus, previously bicolor) is 
unstable, and even on storage in the cold room 
rearranges to a less active compound which 
according to mass spectrometry is isomeric and 
shows a different cracking pattern compatible 
with the assumption of the migration of a 
double bond close to the single nitrogen in the 
molecule. Efforts are being continued to pre- 
pare a crystalline salt or a heavy atom con- 
taining derivative of batrachotoxin or of the 
more stable rearrangement product. One of 
the major obstacles in obtaining suitable 
crystals was the paucity of venom. This has 
now been remedied by our last expedition into 
the Choco jungle of western Colombia, Novem- 
ber 1965 to January 1966. Our collector, Mrs. 
Marte Latham, was able to amass more than 
5,000 frogs (National Geographic Magazine, 
May 1966). Dr. John Daly has been able to im- 
prove the workup of the frog skins and has 
now active material equivalent to 180 mg. of 
crystalline batrachotoxin. 

The Pharmacology and Toxicology of Batra- 
chotoxin 

The pharmacology and toxicology of batra- 
chotoxin has received renewed attention be- 
cause one individual was exposed to the venom 
and has suffered damage described as com- 
parable to a mild case of poliomyelitis. 



Venoms of Other Amphibians 

Dr. John Daly has made a study of frogs 
in Panama, Venezuela, Colombia, and Puerto 
Rico and has noticed that frogs of the genus 
Rana, Brady cephalus, Phyllomedusa, Hyla, 
Atelopus, Eleutherodactylus, Pleurodema, 
Phrynohyas, Prostherapis, and Dendrobates 
contain toxic principles of unknown structure. 
The genera most closely related to Phyllobates 
are Dendrobates and Prostherapis. The exam- 
ination of three species of Prostherapis, one 
additional species of Phyllobates and three 
Dendrobates has now established the absence 
of batrachotoxin, although less active steroidal 
alkaloids may be present. 

The Synthesis and Biosynthesis of Dehydro- 
bufotenine, the Major Indole Metabolite 
from the Parotid Gland of the South 
American Toad, Bufo Marinus 

The structure of the novel tricyclic indole, de- 
hydrobufotenine (cf. Marki, Robertson, and 
Witkop, J. Am. Chem. Soc. 1961) has now 
been confirmed by synthesis. The starting ma- 
terial, 5-benzyloxygramine, is nitrated exclu- 
sively at position 4, converted to the nitrile, 
and, after reduction of the nitro to the amino 
group, hydrolyzed by alkali to the acid and 
cyclized to the 6-membered lactam, which 
could only be reduced to the amine with di- 
borane. The O-benzyl group was hydrogeno- 
lyzed and the resulting biswordehydrobufote- 
nine quarternized to yield dehydrobufotenine. 
Nor- and biswordehydrobofotenine, which have 
also been prepared, are unstable and have not 
been detected in extracts of toad parotid 
glands. Dehydrobufotenine occurs in Bufo 
marinus and Bufo bufo japonicus together 
with the following related 5-hydroxyindoles: 
serotonin, N-methylserotonin, bufotenine, and 
bufotenidine. Tissue slices of the parotid gland 
of Bufo marinus converted tritium-labeled 5- 
hydroxy-DL-tryptophan, serotonin, N-methyl- 
serotonin and bufotenine to dehy drobuf ote- !■ 
nine. Optimal conversions were obtained with' 
tritiated bufotenine. The biosynthetic pathway 
to dehydrobufotenine consists of decarboxyla- [ 
tion of 5-hydroxytryptophan to serotonin,} 
followed by consecutive N-methylations to bu-j 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



219 



fotenine. Finally intramolecular cycle-dehydro- 
genation or oxidative cyclization leads from 
bufotenine to dehydrobufotenine. (S. Senoh 
an J. W. Daly) 

The Reactivity toward N-Bromosuccinimide of 
Tryptophan in Enzymes, Zymogen, and 
Inhibited Enzymes 

The oxidation of tryptophan by N-bromo- 
succinimide (NBS) in a-chymotrypsin, acetyl- 
chmyotrypsin, diisoprophylphosphoryl (DIP)- 
chymotrypsin, N-p-toluenesulfonyl-L-phenyla- 
lanine chloromethyl ketone (TPCK)-inhibited- 
chymotrypsin, and chymotrypsinogen A was 
investigated over a pH range of 4.0-7.0. The 
native enzyme possessed 2-3 more moles of 
NBS-reactive tryptophan at pH 5.5-6.0 than 
the inhibited enzymes or the zymogen. Acetyl- 
chymotrypsin after deacylation behaved 
exactly like native chymotrypsin in its oxidiza- 
bility by NBS. This suggests that the difference 
in reactivity of bound tryptophan is the result 
of a conformational change. Much smaller dif- 
ferences in reactivity were observed in the 
pH range 5.5-6.0 between trypsin, DIP-trypsin, 
and trypsinogen; however, a large difference 
was noticeable between trypsin and its complex 
with the inhibitor from beef pancreas, where 
at pH 5.0, two tryptophan equivalents in the 
complex were protected from oxidation by 
NBS. The pH effect in the reaction of NBS 
with proteins and the use of NBS in effecting 
selective modifications of proteins permits de- 
scription of tertiary structure in chemical 
terms. (T. F. Spande and N. M. Green) 

Novel Photocyclization of Tryptophan Derivcu- 
tives 

Brief irradiation of N-chloroacetyl-L-tryp- 
tophan with a high pressure mercury lamp 
with Vycor filter have the lactam of L-tryp- 
tophan-4-acetic acid, viz., the tricyclic 4-ca- 
roxy-6-oxo,3,4,6,7-tetrahydro-lH,5H-azocine 
[4,5,6-c,d] -indole. The yield in neutral aqueous 
solution exceeds 40%. This reaction makes ac- 
cessible 4-substituted tryptophans and tryp- 
tamines in a one-step procedure and opens up 
new avenues to analogs and homologs of 
dehydrobufotenine, as well as of lysergic acid. 
(O. Yonemitsu and P. Cerutti) 



Photor -eductions of Tryptophan 

UV-irradiation of aqueous solutions of L- 
tryptophan in the presence of excess sodium 
borohydride leads to 4,7-dihydro-L-tryptophan 
(10%), 2,3-dihydro-L-tryptophan (4%), indole- 
3-propionic acid (2%), and a trace of trypta- 
mine. The catalytic reduction of 4,7-dihydro- 
L-tryptophan gives the unstable 4,5,6,7- 
tetrahydro-L-tryptophan, whose racemate was 
obtained by alkaline decarboxylation of 2-car- 
bethoxy-4, 5, 6, 7-tetrahydro-DL-tryptophan, 
ethyl ester. Birch reduction of L-tryptophan 
gave 4,7-dihydro-L-tryptophan, identical with 
the main product of the photoreduction, in 
55% yield. (O. Yonemitsu and P. Cerutti) 

Novel Photocyclization in the Tyrosine and 
Tyramine Series 

N-Chloroacetyl-meta-tyrosine on irradiation 
undergoes photocyclization to the newbicyclic 
7-membered lactam of 3-hydroxyphenylala- 
nine-6-acetic acid. The same photocyclization 
carried out with N-chloroacetyl-tyrosine did 
not lead to identifiable products. However, N- 
chloroacetyl-p-O-methyl-tyrosine gave a novel 
yellow product, X 350 m^, whose structure is 
under investigation. (O. Yonemitsu) 

Photoreduction of Histidine 

By shorter irradiation time the photoreduc- 
tion of histidine can be conducted in such a 
way that the formation of secondary trans- 
formation products is avoided. The acid-labile 
primary photoreduction product has been ob- 
tained in pure form by careful column chrom-- 
atography and has been characterized by 
crystalline benzoyl and tosyl derivatives. The 
mass spectrum of these compounds is sugges- 
tive of dimeric structures. If this observation 
is substantiated by further determinations, the 
photoreductive coupling of two histidines in 
close proximity in a protein or enzyme be- 
comes an interesting possibility. (Y. Fujita) 

The Binding Characteristics of Partially Re- 
duced Polyuridylic Acid 

Selective photoreduction of uridine has now 
been extended to polyuridylic acid. The bind- 



220 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ing capability of poly U/H 2 U was examined 
by means of quantitative infrared spectroscopy 
of aqueous solutions. The interpretation of the 
spectra was based upon previous studies of the 
poly A-poly U system and upon the spectra of 
H 2 Up and other dihydrouracil nucleoside 
model compounds. A comparison of the binding 
characteristics of poly U/H 2 U with poly A 
as a function of the H 2 U content showed that 
the affinity of the two polymers decreases pro- 
gressively with the extent of reduction of poly 
U. (P. Cerutti, H. T. Miles, and J. Frazier) 

Template Activity of Uridyllic Acid — Dihy- 
drouridylic Acid Copolymers 

Dihydrouridylic acid (H 2 Up) has been de- 
scribed as a minor component of alanine-sRNA 
and serine-sRNA from yeast. Polymers con- 
taining various rations of uridylic acid and 
dihydrouridylic acid have been tested for tem- 
plate activity in both the amino acid incor- 
poration system and the binding of aminoacyl- 
sRNA to ribosomes. The results show that dihy- 
drouridine cannot substitute forA,G,C, or U 
in U-containing codons and apparently is not 
recognized in these assays for template ac- 
tivity. (F. Rottman and P. Cerutti) 

Mechanism of the Two-step Reduction of 
Thymidine 

Thymidine in its photoexcited state is re- 
duced by sodium borohydride. The two-step re- 
action involves first photoreduction of the 5,6- 
double bond and, in a subsequent light-inde- 
pendent step, reductive cleavage between posi- 
tions 3 and 4 of the dihydrothymine ring. The 
second reductive step is generally observed 
with dihydropyrimidine nucleosides. The prod- 
uct of dihydrothymine on reductive ring 
opening is 3-ureido-2-methylpropanol-l. The 
structure of this product rests on spectral and 
chemical evidence and on the synthesis by an 
independent route. The mechanism of the pri- 
mary photoreduction was studied by the use 
of differently labeled reducing systems, 
(NaBD 4 /H 2 0; NaBH.,/D 2 0; NaBD 4 /D 2 0) com- 
bined with nmr spectroscopy. The hydro- 
gen donated by NaBH, enters to molecule at 
C-5, whereas the hydrogen added to C-6 



originates from the solvent. (G. Balle and P. 
Cerutti ) 

Stereoisomerism and Stereoinduction in the 
Photoreduction of Thymidine 

Dihydrothymidine has a new optically ac- 
tive center in the 5-position. The abso- 
lute stereochemistry of this new center can 
now be established by cleavage of the ribose 
residue by mild acid treatment and by measur- 
ing the ORD curves of the optically active 
dihydrothymines or the corresponding 3-urei- 
do-2-methylpropanols as obtained by photore- 
duction or catalytic reduction, respectively. (Y. 
Kondo) 

The Stereochemistry of 3-Methylproline 

The known product of Michael condensation 
of crotonaldehyde with diethyl acetamidomal- 
onate can be readily dehydrated to the 
enamide, or converted to the related N-acetyl- 
4,5-dehydro-3-methylproline ethyl ester, which 
was separated into cis and trans forms. These 
diastereoisomers were key intermediates in 
the correlation of cis- and trans-3-methylpro- 
lines with aWoisoleucine and isoleucine. For 
example, the cis enamide N-acetyl-4,5-dehydro- 
cis-3-methyl-DL-proline ethyl ester was con- 
verted, by hydrogenation and hydrolysis, to 
cis-3-methylproline, while its reaction with 
ethyl mercaptan gave a mercaptal which was 
desulfurized to N-acetylalloisoleucine ethyl t 
ester. These correlations confirmed the stereo- 
chemical assignments based upon preferential 
saponification, in which isomeric mixtures of 
N-protected 3-methyl proline esters gave a cis 
ester and a trans acid; the latter procedure 
was also useful for separation of the isomers. 
The NMR spectra of cis- and trans-3-methyl- 
prolines and their derivatives permitted com- 
plete assignments of individual proton peaks. 
By comparison with its published spectrum it 
was confirmed that the 3-methylproline in 
bottromycin A is cis. (A. B. Mauger and F. 
Irreverre) 

Proton Magnetic Resonance Spectra of S,h- 
Dehydroproline Derivatives 

Slight changes in the chemical shifts of pro- 
tons in 3,4-dehydroproline derivatives cause I 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



221 



the appearance of their proton magnetic 
resonance spectra to change markedly, and 
this can be effected by taking one compound in 
two different solvents or by observing closely 
related derivatives in the same solvent. The 
explanation involves a previously undescribed 
type of deceptively simple coupling. A first- 
order analysis of the ABMXX' pattern for 
3,4-dehydroprolinamide in deuterim oxide has 
been made. A full analysis was made of the 
ABXX'and ABXY patterns for this amide in 
deuterium oxide and deuterochloroform, re- 
spectively, after deuterium exchange of the 
labile H 2 ; a very large homoallylic coupling is 
required. Two conformations exist at 40° in 
solution for all N-benzyloxycarbonyl methyl 
esters of proline and its derivatives due to re- 
stricted rotation about the amide bond. Free 
rotation of the amide bond of corresponding 
N-acetyl and N-benzoyl derivatives still 
occured at —50°. The deceptively simple spec- 
trum of N-benzyloxy-carbonyl-2,5-dihydroxy- 
A 3 -pyrroline and that of its diacetate have 
been analyzed as an A 2 X 2 system; a very 
small homoallylic coupling is required. (L. 
Johnson, A. Robertson, and W. Simpson) 

Proline Analogs and Their Effects Upon the 
Biosynthesis ofActinomycins 

Significant differences have been observed in 
the effect of cis- and £rems-4-fiuoro-L-prolines 
on cultures of growing Streptomyces antibio- 
ticus. Unlike proline hydroxylase of chick em- 
bryo the bacterial hydroxylase seems to be 
capable of hydroxylating both cis- and trans- 
ftuoroproline. Considerable radioactivity is 
found in the 4-keto-proline, indicative of loss 
of HF from an unstable intermediary 4-hy- 
droxy-4-fluoro-L-proline. (E. Katz and A. B. 
Mauger) 

Analogs and Homologs of Proline and Hydro- 
xy proline 

An extensive review (277 references) on 
this field of growing importance has been com- 
piled. It is presently serving as a guide for 
attempts to devise inhibitors of proline 
hydroxylase for which the minimal molecular 



weight required is under investigation. (S. 
Udenfriend and A. Mauger) 

The Synthesis of Erythro-yHydroxyl-L-lysine 
and its Nonoccurrence in Collagen 

threo-y-Hydroxy-L-lysine, prepared via y- 
chloro-L-lysine by photochlorination of L-ly- 
sine, was converted to the dicarbobenzyloxy 
lactone, opened to the amide, and oxidized to 
the y-keto derivative. Catalytic hydrogenation 
and debenzylation yielded, after hydrolysis of 
the amide, 72% of the erythro acidlactone mix- 
ture and 28% of the threo pair, which were 
separated by ion-exchange chromatography. 

Catalytic hydrogenation of e-diazo-S-oxo-L- 
norleucine (DON) gave a mixture of 25% 
erythro- and 75% threo-8-hydroxy-L-lysine. By 
reaction with S-methylisothiourea, erythro-y- 
hydroxy-L-homoarginine lactone, the diastere- 
oisomer of the natural threo amino acid from 
Lathyrus, was prepared. Unlike trans-S-hy- 
droxy-L-proline, the position isomer of nat- 
ural 4-hydroxy-L-proline, neither erythro-y- 
hydroxy-L-lysine, the position isomer of 
natural en/£/iro-8-hydroxy-L-lysine, nor its 
threo isomer are regular building stones of 
collagen. (N. Izumiya, Y. Fujita, and F. Ir- 
reverre) 

Synthesis and Metabolism of 6-Hydroxycate- 
cholamines 

The following 6-hydroxycatecholamines (2,- 
4,5-trihydroxyphenethylamines or -phenetha- 
nolamines), potential metabolites of catecho- 
lamines, were synethesized: 3-0-methyl-6-hy- 
droxydopamine, 6-hydroxynorepinephrine, 3-0- 
methyl-6-hydroxynorepinephrine, 6-hydroxye- 
pinephrine, and 3-0-methyl-6-hydroxyepine- 
phrine. Enzymatic O-methylation with catechol 
O-methyl-transferase with S-adenosylmethio- 
nine- 14 C as donor of 14 CH 3 gave radio-active 
14 C-labeled3-0-methyl-6-hydroxycatecholamines 
which were used for metabolic studies in the 
rat. The corresponding phenylacetic and man- 
delic acids, as well as the phenylglyclo, were 
identified as metabolites by comparison with 
synthetic compounds. Eleven new potential 
metabolites were compared and characterized. 
The relative substrate activity of some of 
these 3-0-methyl-6-hydroxycatecholamines with 






222 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



monoamine oxidase was much lower than that 
of 3-O-methyldopamine or normetanephrine. 
(J. W. Daly, J. Benigni, R. Minnis, and Y. 
Kanaoka) 

The Chemorelease of Norepinephrine from 
Mouse Hearts. Structure- Activity Rela- 
tionships. Sympathomimetic and Related 
Amines 

A rapid method for determining the chem- 
orelease of cardiac norepinephrine in the 
mouse has been developed. Endogenous cardiac 
norepinephrine is prelabeled with 5 /xcuries 
of norepinephrine-7- 3 H. The cardiac norepine- 
phrine- 3 H remaining in the heart after 3 
hours averages 340 m^curies. Lower levels of 
radioactivity are found in heart when animals 
are injected 1 hour after the norepinephrine- 
3 H with a compound that causes chemorelease. 
The releasing potencies of a wide variety of 
sympathomimetic amines and related com- 
pounds have been determined. Structure-ac- 
tivity correlations have been established. (J. 
W. Daly and C. R. Creveling) 

Drugs Affecting the Sympathetic and central 
Nervous Systems 

Endogenous cardiac norepinephrine in mice 
has been prelabeled with a 5-/xCurie injection 
of norepinephrine- 3 !!, and the effect of vari- 
ous classes of compounds on the normal 
physiological depletion of norepinephrine- 3 !! 
has been studied. The effect of a variety of 
tranquilizers, antidepressants, ganglionic 
blocking agents, hypotensive agents, sympa- 
tholytics, and compounds that inhibit key 
enzymes in the biogenesis and metabolism of 
norepinephrine have been ascertained. The re- 
leasing and release-inhibiting activities of 
close to 100 representatives of these drugs 
have been measured. (J. W. Daly and C. R. 
Creveling) 

The Depletion of Nor epinephrine- 3 H from 
Heart by a-Methyl-m-tyrosine. A Novel 
and Convenient Method for Assaying the 
Inhibition of Aromatic Amino Acid De- 
carboxylase in vivo 

Cardiac norepinephrine of mice was prela- 
beled with 5 ^curies of norepinephrine- 3 !!. 



After 1 hour 10 mg/kg of a-methyl-ra-tyro- 
sine was administered subcutaneously. After 
3 hours the activity of cardiac norepinephrine- 
3 H was 50% of that of control animals. This 
release of norepinephrine which requires en- 
zymatic decarboxylation of a-methyl-m-tyro- 
sine to the active releasing agent a-methyl- 
m-tyramine is blocked by inhibitors of aro- 
matic amino acid decarboxylase. The resulting 
decrease in a-methyl-m-tyrosine-releasing ac- 1 
tivity is a direct measure of inhibition of the 
enzyme in vivo and provides a convenient 
method for determining the effectiveness of 1 
decarboxylase inhibitors in intact animals. (C. 
R. Creveling and J. W. Daly) 

Preparation of Gramicidin A, B, and C 

By countercurrent distribution and redis- 
tribution the commercially available peptide 
antibiotic, gramicidin, was resolved on a prep- 
arative scale. The group of lipophilic peptides 
contained gramicidins A, B, and C, each of 
which consisted of a pair of congeners, i.e., 
valine-gramicidin (80-95%) and an isoleu- 
cine-gramicidin (5-20%). The sum of aro- 
matic amino acids was always four, namely, 
four tryptophan in gramicidin A, three tryp- 
tophan plus one phenylalanine in gramicidin 
B, and three tryptophan plus one tyrosine in 
gramicidn C. A more hydrophilic, strongly an- 
tibiotic group of peptides, designated grami- 
cidin D, contained five to six additional amino 
acids. Complete amino analyses, including 
time-dependence studies of hydrolyses, have 
been carried out over the entire range of 999 
transfers. (E. Gross) 

Release of Kinin Activity from Human Kinino- 
gens and Fresh Plasma by Cyanogen 
Bromide 

The two human kininogens I and II have 
been treated with cyanogen bromide, a rea- 
gent which under acidic conditions selectively 
cleaves methionyl bonds in peptides and pro-! 
teins. Kinin activities were estimated by the 
muscle contraction of estric rat uterus. As a 
control the stability and recovery of kallidini 
were tested under the same experimental con-i 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



223 



ditions as were used for cleavage of the Kinino- 
gens. In a series of experiments 90-98% of 
the kallidin activity was recovered. Solutions 
of 100 A of Kg I and Kg II, respectively, in 
1 ml of 0.25 N hydrochloric acid containing 
1 mg of cyanogen bromide were incubated at 
35° for 20 hours. The reaction products were 
then lyophilized and assayed for kinin activ- 
ity. Unfractionated native human plasma with 
cyanogen bromide releases kinin activity to 
the extent of 10% of the activity observed after 
treatment with urinary human kallikrein. 





Kinin Activity 

(expressed as y kallidin/mg 

protein) 


Kininogen 


after cleavage 
with CNBr 


after cleavage 

with human 

urinary 

kallikrein 


Kg I 

Kg II 


2.5 
0.8 


8 
6 



The methionyllysylbradykinin (1-11) is pic- 
tured as a unit within the polypeptide chain 
of the precursor protein: 



BrCN BrCN 

Met Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Met— 

1 2 3 4 5 6 7 



(E. Axen, J. V. Pierce, and M. E. Webster). 

Selective Cleavage of Cysteine Peptide Bonds 

Peptides which contain N-alkylated residues 
of cysteine were synthesized. Cleavage of the 
aminoacyl bond of cysteine in model peptides 
is as high as 90-100%. The cleavage of S-al- 
kylcysteine-containing peptides proceeds via 
two different mechanisms: (a) at low tem- 
peratures an oxazolinium bromide is formed. 
Hydrolysis leads to the formation of the-O-acyl 
derivatives which have to be hydrolyzed under 
conditions which prevent an ON-acyl shift 
from taking place, (b) At high temperatures 
/3-elimination is predominant. Water is added 
to the acrylic acid derivative. Hydrolysis re- 
sults in the formation of pyruvic acid and the 
amide of the amino acid that preceded cys- 
teine. 

Cleavage of Cysteine Peptide Bonds in Insulin 
and Ribonuclease 

The disulfide bonds of insulin were reduced 
by treatment of the hormone with dithioery- 
thritol. Various alkyl halides were employed to 
alkylate the liberated mercapto groups. The 
methylated chains of insulin are poorly soluble 
in aqueous solution. Presently the most prom- 
ising alkylating reagent appears to be amino- 
ethylbromide. Yields of conversion of S-alkyl- 
cysteine amount to 75%. 

The S-protein of bovine pancreatic ribonu- 
clease was cleaved with cyanogen bromide. 



9 10 11 12 13 14 15 16 

Liberated homoserine lactone was removed by 
gel filtration. The disulfide bonds of the result- 
ing product were reduced with dithioerythri- 
tol. The mercapto groups were methylated by 
treatment of the reaction mixture with methyl 
iodide and the fragments separated by gel fil- 
tration. The NH 2 -terminal fragment (one resi- 
due of S-methylcysteine) was cleaved with 
cyanogen bromide. The amino acid analysis 
was low in serine indicating that one residue 
of serine has been removed in part with the 
S-peptide. 

Antibiotic Nisin 

The peptide antibiotic nisin is rich in sul- 
fur-containing amino acids: methionine, lan- 
thionine, and /?-methyllanthionine. Little is 
known about the structure of the antibiotic. 
The high thioether content makes the peptide 
a candidate for fragmentation via the cyano- 
gen bromide reaction. A commercial product 
called Nisaplin contains approximately 2% of 
nisen. From this material partially purified 
nisin has been obtained and analyzed. A study 
of the elution pattern of the lanthionines on 
alayzer columns has led to the separation of 
three optical isomers of synthetic /?-methyll- 
anthionine. 

Active Carboxy Group of Pepsin 

Pepsin contains only three amino acids, the 
peptide bonds of which can be cleaved spe- 
cifically with an enzyme. Selective nonenzy- 



224 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



matic cleavage appears to be required to es- 
tablish the primary structure of the enzyme. 
Pepsin was reduced with dithioerythritol and 
carboxymethylated. Treatment of CM-pepsin 
with cyanogen bromide converted all resi- 
dues of methionine to homoserine. Reduced 
pepsin was also methylated and then treated 
with cyanogen bromide. Seventy-five percent 
of the residues of S-methylserine were con- 
verted to serine. p-Bromophenacyl bromide has 
been reported to inactivate pepsin reversibly. 
The proposed involvement of disulfide bond 
was ruled out. By carrying pepsin through a 
series of chemical events it was shown that 
the enzyme contains one residue of active as- 
partic acid: p-bromophenacyl bromide esteri- 
fies the /3-carboxyl group of the active aspartyl 
residue. (E. Gross and J. L. Morell) 

Improvements in the Methodology of Amino 
Acid Analysis 

The first automatic integrator had been de- 
signed and was built prior to the advent of 
accelerated systems for amino acid analysis. 
The threshold-type detection to start and stop 
integration is not applicable to accelerated sys- 
tems. The integrator has been modified by 
adding a second type detection. The slopes of 
a peak that represents a solute which is eluted 
from a column are now monitored. This type 
of ON-OFF control of integration is fully 
satisfactory for the acelerated analytical sys- 
tems. The automatic regeneration of analytical 
columns has been improved and extended to 
additional systems. A dual valve system has 
been designed to add automatically samples to 
the top of columns. The sample has to reach 
the resin without being diluted. This is accom- 
plished by a special, plunger-type connector 
which is in direct contact with the resin. The 
"Robot Chemist" ninhydrin analyzer has been 
modified to handle twice the number of ninhy- 
drin analyses per day. The computer program 
for the calculation of the amino acid composi- 
tion of peptides and proteins has been revised. 
Details, such as minimum molecular weights 
per residue, numbers of individual residues, 
and molecular weight calculations are now 
readily available. (E. Gross and J. L. Morell) 



LABORATORY OF EXPERIMENTAL 
PATHOLOGY 

Introduction 

In July, 1965, Dr. Benjamin Highman, 
Chief, Section on Pathologic Anatomy, was 
assigned as PHS Liaison Officer to the Armed 
Forces Institute of Pathology (AFIP) and was 
appointed Chief of the Institute's Radiopathol- 
ogy Division "I." He also remained a member 
of the staff of the Laboratory of Experimental 
Pathology, NIAMD, continuing with research 
projects active at the time of his assignment 
to the AFIP. 

The duties of the Liaison Officer are varied. 
He serves on the Intra-Mural Interagency Ad- 
visory Group of the AFIP, which meets weekly. 
He keeps the pathologists of the PHS informed 
about the various courses, educational aids, 
and other activities of the AFIP that may be of 
interest to them. In general, he is concerned 
with matters of mutual interest to the PHS 
and the AFIP. 

As Chief of the Radiopathology Division "I,' 
he takes part in and has administrative respon- 
sibilities for the work of the Radiologic Path- 
ology Branch and the Radiation Pathology 
Branch. He participates in the educational pro- 
gram for radiology residents given by the Ra- 
diologic Pathology Branch; he usually devotes 
several hours monthly in giving lectures cor- 
relating the radiologic and pathologic pictures. 
He has also undertaken several research proj- 
ects related to radiation pathology. 

Following Dr. Highman's assignment, the ac- 
tivities of the Section were handled by the 
Chief of the Laboratory. Assistance to the Di- 
vision of Indian Health, PHS, the Hospital of 
the Coast Guard Academy, New London, and 
to several Federal Prisons in the form of path- 
ologic consultations and examination of tis- 
sues submitted for diagnosis has continued un- 
altered. The Laboratory staff trained in path- 
ology was greatly helped through voluntary and 
generous participation in this activity by Dr. 
Ruth Kirschstein (Chief of Pathology, DBS) 
and Dr. Richard Asofsky (LGAR-NIAID) 

In its research activities, the Laboratory 
staff has made increasing use of the available 
electron microscopes, a trend expected to con- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



225 



tinue. It necessitated a larger staff of techni- 
cal assistants in the preparation of material. 
The additional use of bacterial and tissue cul- 
ture systems in investigations by other mem- 
bers has made it possible to utilize positions 
previously used in the general tissue labora- 
tory for these special purposes. For the same 
reason, a reduction in the preparation of mi- 
croscopic slides for investigators outside the 
Laboratory and the Institute became necessary. 
A satisfactory solution to this problem was 
found when the outside laboratories agreed to 
provide personnel and our Laboratory training 
and technical equipment to handle such ma- 
terial. 

The Laboratory is host to two foreign guest 
workers, Dr. Iwao Hirono and Dr. Nestor Piva. 
Dr. Hirono, Associate Professor of Pathology 
at Gifu University, is the holder of an Eleanor 
Roosevelt International Cancer Fellowship; Dr. 
Piva,- Head of the Department of Pathology, 
in the Institute for Experimental Medicine, 
Aracaju-Sergipe-Brazil, is sponsored by the 
Brazilian Government. Dr. Hirono's work is 
concerned with studies of carcinogenetic effects 
of cycasin and Dr. Piva's area of interest con- 
cerns special problems related to the histo- 
chemistry of schistosomes. 

Pathology of Rheumatic Diseases 

The elasticity of aging human cartilage, ob- 
tained at necropsy, was studied by an inden- 
tation technique. Costal cartilage was stiffer 
than articular tissue at all ages. The Young's 
modulus of rib cartilage was greater in middle 
aged than young adults; by contract the values 
for patellar cartilage were not significantly 
different in the two age groups. Nor was the 
recovery from deformation diminished. In os- 
teoarthritic fibrillation, there was a marked 
softening as well as a measurable unrecovered 
strain. These observations contradict a widely 
held, undocumented view that senescent im- 
pairment of the elasticity of articular cartilage 
is a predisposing factor in the development of 
degenerative joint disease. (Dr. Sokoloff) 

Motion of the embryonic limb was found 
necessary for the proper development of ani- 



mal joints. Movement was prevented in chicks, 
beginning of the seventh day of incubation, by 
three techniques: administration of botulinum 
toxin, decamethonium chloride, or extirpation 
of the lumbosacral spinal cord. Although pri- 
mordial development of the joint anlage pro- 
ceeded to a certain point, the joint cavity and 
certain accessory articular structures, such as 
the cruciate ligaments and some sesamoid 
bones, failed to develop. Variable cartilagin- 
ous ankylosis ultimated supervened. (Dr. 
Drachman [Pratt Institute, Boston, Mass.] 
and Dr. Sokoloff) 

It was shown that, over short time inter- 
vals, the friction of cartilage sliding against 
glass is nearly unaffected by changes in load, 
as would be expected for a weeping bearing. 

A theoretical investigation has shown that 
lubrication of bearings by osmotic type forces 
arising from adsorbed layers of long chain 
polymer molecules (like synovial mucin) is 
thermodynamically possible. (Dr. McCutchen) 

Animal Joint Lubrication 

The coefficient of friction during sliding can 
best be analyzed by breaking it down into its 
components, namely: viscosity of the lubricant, 
incremental deformation or plowing, and ad- 
hesion. In a dogs ankle, fluid viscosity had little 
or no affect on friction loss; plowing was ap- 
preciable at low speeds and decreased with in- 
creasing speed; adhesion was much lower with 
synovial fluid than other lubricants due to a 
lubricity producing factor in it. The coefficient 
of friction decreased with increasing loads at 
constant speed. The coefficient of friction was 
low in the joint since the load was mainly car- 
ried by self -pressurized fluid in the water rich 
articular cartilage. The friction losses during 
dynamic or cyclic loading, similar to that pro- 
duced when walking, were comparable to those 
obtained with constant loads. The addition of 
hyaluronidase to synovial fluid did not change 
the lubricating quality of synovial fluid. Puri- 
fied hyaluronic acid in an isotonic saline solu- 
tion gave a friction loss similar to the saline so- 
lution and 2.5 times that obtained with syno- 
vial fluid. (Mr. Linn) 



226 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Cytogenetics 

Experiments on Reutilization of Chromosomal 
DNA by Cells Grown In Vitro 

This project is an attempt to determine 
whether cells in culture will reutilize foreign 
DNA in the form of isolated single chromo- 
somes. Chinese hamster fibroblast cultures 
labeled with H 3 . The metaphase cells were 
separated, fragmented and the chromosomes 
isolated. These were then inoculated into ham- 
ster cell cultures and human blood cultures. 
Autoradiographic preparations obtained from 
these cultures showed weak labeling on several 
chromosomes in many mitosis. In one experi- 
ment, which requires confirmation, no labeling 
was detected when the cell culture was given a 
large excess of unlabeled base (thymidine). 
Thus, it seems that fibroblasts and lymphocytes 
growing in vitro reutilize foreign chromosom- 
al DNA. Tentative findings indicate that this 
DNA is first digested and the constituents en- 
ter the cells' metabolic pool of purines, pyrmi- 
dine, etc. This appears to be relevant to recent 
claims that animals can incorporate foreign 
nucleic acids into their systems. (Dr. Tjio) 

Meiotic Chromosome Studies in the Human 
Male 

In the meiotic prophase the chromosome is 
differentiated into morphologically distinct 
regions (chromomeres) which are constant in 
size and position. These regions have been de- 
scribed and correlated with certain genotypic 
expressions in many organisms, especially in 
corn (Zea mays) of which elaborate gene 
maps have been obtained. 

This study is aimed at obtaining similar 
gene maps of the human male meiotic pro- 
phase, in particular the pachynema. Experi- 
ments are being carried out to obtain well pre- 
served pachytene chromosome preparations 
suitable for mapping the structural morphol- 
ogy. Human testicular biopsy material has 
been provided by the Surgical Department of 
the George Washington Hospital. (Dr. Tjio) 

Characterization and Quantitation of Enzymes 
in Histomchemical Systems 

A continuing study of the characteristics of 



hydrolytic enzymes demonstrated using histo- 
chemical substrates in both histochemical and 
biochemical systems has resulted in the ideni- 
fication of a wide variety of hitherto unde- 
scribed aminopeptidases residing predominant- 
ly in the particulate fraction of the cell. 
Aminopeptidase B, an exopeptidase selective 
for N-terminal arginine and lysine residues of 
peptides and polypeptides, has been highly 
purified (over 1000 fold) from rat liver and 
shown to be the first described peptidase acti- 
vated by an anion (Cl~). This enzyme is ca- 
pable of catalyzing the conversion of Kalledin 
X to Bradykinin without further degradation 
of the nonopeptide. The possible significance of 
cellular anionic changes in potentiating the 
formation of Bradykinin are under investi- 
gation. 

In addition to the previous finding of a Co ++ - 
activated aminopolypeptidase from rat and hog 
kidney, a new class of enzymes acting on di- 
peptide naphthylamides (e.g. Ala-Ala-/3-naph- 
thylamide) with a high degree of substrate 
specificity have been found. These enzymes do 
not attack the N-terminal amino acid, but have 
a selectivity for the N-terminal dipeptide mo- 
iety. None of the members of this group have 
been found to be anion or cation activated. 
Efforts to characterize enzymes acting on the 
histochemical substrates acetyl- and a-halo- 
genated acetylnaphthylamides have resulted in 
the identification of kynurenine formamidase 
as one of the chief effectors of the hydrolytic 
reaction in rat tissue homogenates (liver and 
kidney). Other as yet poorly-defined enzymes 
also appear to be implicated in the hydrolysis 
of this class of substrates. (Dr. Glenner and 
Drs. Vanha-Perttula and Hopsu [Turku Uni- 
versity, Turku, Finland] ) 

Histochemical Localization of ATPase Activity 

Studies on the lead-salt method of Wachstein 
and Meisel for localization of tissue ATPase 
activity have confirmed previous reports by 
other investigators that both fixation and the 
presence of lead ions markedly inhibit tissue 
ATPase activity as measured by biochemical 
techniques. Further studies have demonstrated 
that lead alone may catalyze the nonenzymatic 
hydrolysis of ATP under conditions employed 



NATIONAL INSTITUTE OF AKTHRITIS AND METABOLIC DISEASES 



227 



for the Wachstein-Meisel procedure. In addi- 
tion, localization of lead deposits to plasma 
membranes occurred only with those lead and 
ATP concentrations which gave significant 
nonenzymatic breakdown of ATP by lead. 
These studies indicate that localization of lead 
deposits to specific tissue sites does not con- 
stitute sufficient evidence for the presence of 
ATPase activity in those sites. (Dr. Moses and 
Drs. Rosenthal and Beaver [Vanderbilt Uni- 
versity, Nashville, Tenn.] ) 

Studies in Oxidative Phosphorylation 

A derangement of oxidative phosphorylation 
may be a factor contributing to the elevated 
serum enzyme levels observed after animals 
are subjected to stress. Studies of the mechan- 
isms by which chemical reagents depress this 
function in mitochondria had shown that these 
compounds are bound to the protein of the 
organelle. It was demonstrated that mitochon- 
dria containing the bound uncoupling reagents 
were more susceptible to the attack of proteo- 
lytic and lipolytic enzymes and this may be a 
factor in the cellular pathology of stress. It 
was shown that serum albumin not only could 
restore oxidative phosphorylation to chemi- 
cally damaged mitochondria, but could also 
rapidly restore the more sensitive parameter, 
respiratory control. Electron microscopy re- 
vealed that serum albumin also prevented the 
morphological deterioration induced by the un- 
coupling reagents. Cellular and serum proteins 
may play a similar role in the recovery of 
stressed animals. (Dr. Garbus and Dr. Wein- 
bach [LPD-NIAID] ) 

Mechanism of Protection by 5-HT in Tryptophan 
Deficiency 

It had previously been noted in a study of 
experimentally induced cardiac lesions of the 
carcinoid type in guinea pigs that 5-hydroxy- 
tryptamine (HT) reduced the incidence of my- 
ocardial lesions as well as the mortality of the 
tryptophan deficient animals. The "protective 
effect" of 5-HT was limited to the myocardium 
and had no influence on the endocardial altera- 
tions in these animals. In an attempt to evalu- 
ate the pathogenesis of the myocardial lesions 



experiments were performed to study changes 
in the myocardium prior to microscopically 
visible injury. 

It was found that a tryptophan deficient diet 
in guinea pigs produced a decrease in total 
phosphorylase and that this depression was 
prevented completely by exogenous 5-HT and 
in part by supplementing the deficient diet 
with DL-tryptophan. The protective effect of 
5-HT appeared to be time dependent since it 
was no longer seen after tryptophan deficiency 
was established. 

The mechanism by which 5-HT protects the 
mycardium of guinea pigs on a tryptophan 
deficient diet is still uncertain although the re- 
sults of the experiments indicated that 5-HT 
increased phosphorylase a activity by convert- 
ing phosphorylase b to a since total phos- 
phorylase was not altered. The essentially sim- 
ilar effect of exogenous 5-HT on reserpinized 
and nonreserpinized guinea pigs in increasing 
phosphorylase a activity showed that this ef- 
fect was still obtained after depletion of endo- 
genous 5-HT and norepinephrine following 
reserpine injection. When comparing the in- 
creases in phosphorylase after 5-HT adminis- 
tration between the two groups, the hearts of 
nonreserpinized animals showed a greater in- 
crease in the percentage of conversion of phos- 
phorylase b to a. Whether the slightly lower 
response of reserpinized animals is due to the 
formation of abnormal binding centers for 
5-HT as suggested by E. Kuntzman, et at. 
(Fsd. Proc. 15: U50, 1956) is unknown. (Dr. 
Spatz) 

Fluorescein Studies 

Localization of Antigens in Tissues 

During the past year, studies involving the 
localization of endogenous antigenic substances 
by means of specific fluorescent antibody have 
been extended to include the enzyme ^glycero- 
phosphate dehydrogenase in the heart muscle 
of the rat where it is distributed in mitochon- 
dria surrounding the fiber. This distribution 
has been shown in relation to the A band of 
the muscle fiber which is identified by stain- 
ing with fluorescent anti-myosin globulin. The 
distribution of the enzyme is similar to that 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of glyceraldehyde phosphate dehydrogenase 
with which it is closely linked in the glyco- 
lytic cycle of the msucle metabolism. (Dr. Em- 
mart) 

Localization of prolactin in rat pituitary and 
in a transplantable mammotropic pituitary 
tumor using fluorescent antibody was com- 
pleted demonstrating the presence and dis- 
tribution of prolactin in acidophilic cells of 
tumor tissue, especially in those cells contain- 
ing erythrosiniphilic granules. Bioassay of the 
isolated rat tumor prolactin indicates that it 
contains 0.15 International units per mg. Pre- 
cipitin reactions in the test tubes show that it 
precipitates readily with antibody to ovine 
prolactin. A comparison of the electrophoretic 
pattern of ovine prolactin and rat tumor prolac- 
tin shows that they have different rates of 
migration and that the tumor prolactin con- 
tains some impurities. (Dr. Emmart and Mr. 
Turner and Dr. Bates [LNE-NIAMD] ) 

Localization of Prolactin in Fundulus Hetero- 
clitus, L. 

It has been established that antiovine pro- 
lactin will precipitate with fish prolactin. This 
antibody has therefore been used to localize 
prolactin in erythrosinophilic cells of the ros- 
tral pars distalis of the teleost, F. heteroclitus, 
L. Further studies in immunochemistry of fish 
prolactins are in progress. (Dr. Emmart and 
Dr. Pickford [Yale University, New Haven, 
Conn.] and Dr. Wilhelmi [Emory University, 
Atlanta, Ga.] ) 

Studies on Nucleic Acids and Protein Synthesis 

Autoradiographic and cytochemical studies 
regarding structure and function of the nu- 
cleolus in relation to nucleic acid and nucleo- 
protein synthesis have been continued. In or- 
der to develop suitable methods for electron 
microscopic investigation of nuclear and nu- 
cleolar ultrastructure, a study of the effect of 
fixation on the autoradiographic localization 
of nucleoprotein synthesis in nucleoli was un- 
dertaken. The effect of temperature and pH of 
formaldehyde, gultaraldehyde and of acid al- 
cohol fixation on the localization of HMysine, 
H 3 -arginine and H 3 -uridine was studied in 



PPLO-free HeLa cells under conditions of se- 
lective inhibition of nucleolar RNA synthesis 
Actinomycin D and of ribosomal protein syn- 
thesis with Puromycin. It was found that the 
temperature of fixation with P0 4 buffered for- 
maldehyde and glutaraldehyde alters the intra- 
nuclear distribution of HMysine and H 3 - 
arginine after brief labeling, without apparent 
loss of nuclear uptake. Fixation at 37° C, as 
compared with 0° or 23° C, results in a shift 
to chromatin of about 25% of the ratio of nu- 
cleolar to nuclear grain count/unit area (a 
<0.01). A similar redistribution was found 
when cells were formalin-fixed at acid pH, with 
acetic rethanol (Carnoy), or were eluted after 
fixation with hypertonic alkaline Tris buffer. 
This shift of nucleolar label appears to in- 
volve ribosomal protein, since a proportional 
inhibition, with loss of nuclear uptake, occurs 
after treatment with Puromycin (200 mgm/ 
ml) or with Actinomycin D (0.05 mgm/ml). 
In the former case inhibition occurs under con- 
ditions not inhibitory to nucleolar RNA syn- 
thesis, but in the case of Actinomycin there is 
a parallel selective inhibition of nucleolar 
RNA, shown by R. P. Perry (NCI Monograph 
No. 14, 73-91, 1964) and others to be a riboso- 
mal precursor. By removing the inhibitor, the 
effect of Puromycin can be reversed only where 
cells are fixed at 0° or 23° C. These findings, 
together with previously reported data, are in- 
terpreted as evidence for the rapid incorpora- 
tion in the nucleolus of amino acid label into 
a basic ribonucleoprotein participating in the 
synthesis or stabilization of the ribosome 
complex. This ribonucleic protein unstable un- 
der some conditions of fixation previously em- 
ployed and solubilized in aldehyde fixative at 
slightly elevated temperature probably as a 
result of thermal denaturation of associated 
RNA. (Dr. Suskind) 

An autoradiographic and virologic study, in 
collaboration with Dr. G. Rabotti (LVO- 
NCI) designed to investigate the sites of viral 
nucleic acid synthesis and viral protein syn- 
thesis of Rous sarcoma virus in transformed 
(neoplastic) and nontransformed chicken fi- 
broblast cultures is in progress. Preliminary 
results point to a nuclear site of viral nucleic 
acid synthesis. (D. Suskind) 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



229 



Studies on Soluble Ribonucleic Acid (sRNA) 

A new method for the chromatographic 
fractionation of different sRNA species has 
been developed. A gradient of increasing salt 
and pH is employed to elute sRNA from 
DEAE-Sephadex. There is evidence that reso- 
lution is dependent upon a change in the 
sRNA from a partially denatured state at the 
beginning of elution to the native state at the 
end. The method has a large input capacity 
and provides good resolution of certain sRNA 
species. It is convenient to use and can be 
adapted either for preparative or analytical 
work. (Dr. Smith) 

Electron Microscope Studies 

Studies in Amyloidosis 

Electron microscopic evidence has indicated 
the morphologic identity of amyloid fibers ob- 
tained from both the primary and secondary 
disease. Recent electron microscopic studies on 
negatively stained extracts from human aray- 
loidotic tissue revealed the presence of two 
distinct types of particles. One was a 100 A 
wide rod, while the other was a small penta- 
gonal structure (unit structure) 90 A in diam- 
eter both morphologic units were present in 
all preparations obtained from various tissues 
of patients with either primary or secondary 
amyloidosis. Narrow electron dense bands di- 
vided the rods at intervals into smaller seg- 
ments with the average distance from the cen- 
ter of one segment to the center of the next 
being approximately 40 A. Frequently, how- 
ever, the width of the electron dense bands be- 
tween segments varied irregularly, suggesting 
that the segments were easily disunited. When 
preparations containing predominantly long 
rods were sonicated, a large increase in the 
number of unit structures was evident. The 
unit structures seemed to be single or double 
segments of rods lying on their flat side, thus 
affording an end view of the amyloid rod. They 
appeared to be composed of five globular sec- 
tions surrounding a hollow core. Purification 
of this periodic component of amyloid to afford 
a single protein band on disc electrophoresis 
and reconstitution of the rod has been 



achieved. (Dr. Glenner and Dr. Bladen [LHP- 
NIDR]) 

Fine Structure and Cytochemistry of Rabbit 
Marrow Cells 

A descriptive analysis of granulocyte devel- 
ment and of the origin, nature, and fate of 
their characteristic granules (especially those 
of the heterophil); localization of acid and 
alkaline phosphatases in this tissue. 

The mode of eosinophil granules formation 
has been detailed within the past year and 
incorporated into this completed study. Small 
vacuoles of moderately dense, flocculent con- 
tents arise from Golgi lamellae. Apparently 
the contents of these vacuoles condense cen- 
trally, resulting in the characteristically dense 
and homogeneous substance of eosinophil gran- 
ules. Fusion of additional vacuoles and further 
condensation result in large spherical gran- 
ules which mature individually, acquiring the 
angular profile and included crystalloids which 
are typical of eosinophil granules in this spe- 
cies. (Drs. Wetzel and Spicer and Dr. Horn 
[Vanderbilt University, 'Nashville, Tenn.] ) 

Myeloid Elements 

Investigation into the fine structural aspects 
of granulocyte development and cytochemis- 
try have been extended to include investigation 
of human granulocytes in health and disease. 
Phosphatase cytochemical methods, ultrastruc- 
tural demonstration of mucosubstances and 
electron microscope autoradiography with 
S 35 4 = and with tritiated arginine have been 
utilized to characterize leukocyte granules of 
humans and experimental animals. (Drs. Wet- 
zel and Spicer and Dr. Horn [Vanderbilt Uni- 
versity, Nashville, Tenn.] ) 

Studies of Human Lymphocytes after Exposure 
to Phytomitogens 

In vitro transformation of human peripheral 
small lymphocytes with phytomitogens has 
been studied. The physiochemical, biologic and 
morphologic features of cells stimulated with 
pokeweed mitogen (PWM), an extract of 
Phytolacca americana reveals that this mate- 
rial has properties which differ from phyto- 



230 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



hemagglutinin (PHA). Poke weed mitogen 
stimulation is maximal after 72 hours in cul- 
ture. With optimal concentrations of PWM 
50-60% of the initial cell inoculum transforms. 
At 72 hours the cell population is comprised of 
small- and medium-sized lymphocytes, large 
PHA-like blast cells, and a unique intermedi- 
ate-sized cell type. The fine structural features 
of these intermediate cells include an eccen- 
tric nucleus, with clumped heterochromatin, a 
well developed Golgi apparatus, ribosomal ag- 
gregates, and a well developed rough-surfaced 
endoplasmic reticulum. These fine structural 
features differ importantly from those con- 
sistently seen in PHA transformed cells, and 
are comparable to those of cells which have 
been described as early antibody-producing 
cells. (Dr. Douglas, Drs. Chessin and Borjeson 
[LI-NIAID] , and Mr. Hoffman) 

Fine Structural Characterization of 
Neuromelanin 

Neuromelanin 

Electron microscopic and cytochemical 
and rhesus monkey substantia nigra and locus 
coeruleus have been concluded. Melanin and 
other pigments from various anatomical sites 
were also examined for the purpose of compar- 
ison with the neuromelanin pigment. A sub- 
structure different from that of other melanin 
pigments was found in neuromelanin which 
more closely resembled neuronal lipofuscin pig- 
ment. Neuromelanin granules, however, con- 
tained a reducing substance in the form of a 
very electron dense component — a structure 
not found in neuronal lipofuscin pigment gran- 
ules. (Dr. Moses and Drs. Ganote and Beaver 
[Vanderbilt University, Nashville, Tenn.] ) 

Experimental Porphyria 

Allylisopropylacetamide (AIA) induced por- 
phyria, in rats is characterized by hepatome- 
galy and increased production in liver of the 
porphyrin precursors, -aminolevulinic acid 
and porphobilinogen. The latter has been 
shown to be secondary to induction of liver 
8-aminolevulinic acid synthetase, the rate-lim- 
iting enzyme of heme biosynthesis. Light and 



electron microscopic studies on this experi- 
mental model, which has been extensively in- 
vestigated biochemically, revealed marked hy- 
pertrophy of hepatic parenchymal cells and 
little evidence of cellular proliferation. Elec- 
tron microscopically the enlarged hepatic cells 
exhibited increased numbers of secretory vesi- 
cles, extensive development of the smooth en- 
doplasmic reticulum, and striking nucleolar 
enlargement — changes which can probably be 
explained on the basis of increased secretory 
activity, drug detoxification and enzyme induc- 
tion with increased protein synthesis. Both the 
morphologic and metabolic effects of AIR were 
blocked by the simultaneous administration of 
either Actinomycin D or Puromycin. A com- 
bined biochemical electron microscopic and 
autoradiographic study of amino acid and 
nucleoloid uptake under these conditions is in 
progress. (Drs. Moses and Suskind and Dr. 
Tschudy [ODir-NCI] ) 

Cytochemistry of Bacterial Phosphatases 

The localization of several phosphatases in 
E. coli has been studied in an attempt to con- 
firm and extend biochemical findings in this 
system. Substrate specificity, genetic differ- 
ences among various strains, and pH optima 
have been used experimentally to distinguish 
among the enzymes. Selective localization of 
cyclic diesterase and glucose-6-phosphatase to 
the space between the cell wall and the plasma 
membrane, often forming prominent polar 
caps, is of particular interest in view of (1) 
the lack of significance attached to this region, 
and (2) recent data on the selective permea- 
bility of the cell wall. This region may thus 
serve to compartmentalize metabolic events in 
a manner analogous to intracytoplasmic mem- 
brane-limited cisternae in metazoan cells. An 
alkaline phosphatase appears to be localized 
primarily to the surface of these organisms, 
but a pH-dependent penetration factor has not 
been excluded. (Drs. Wetzel and Spicer, and 
Dr. Heppel [LBM-NIAMD] ) 

Temperature Effect on Lipids of Leishmania 

Although primarily a biocehmical analysis of 
shifts in fatty acid composition at elevated 
culture temperature, the initial identification 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



231 



of cytoplasmic droplets as lipid and supportive 
morphological evidence was provided by this 
Laboratory. (Dr. Greenblatt [LPB-NIAMD] 
and Dr. Wetzel) 

Mouse Thyroid Gland Cytology 

This is a descriptive morphological study of 
this organ; unique features include ciliated 
follicular epithelia, several unusual types of 
follicular cell, and the elaboration of acid mu- 
cosubstance. (Dr. Wetzel and Dr. Wollman 
[LPHY-NCI] ) 

Thyroid Parafollicular {Light) Cells 

Preliminary histological findings in long-term 
(16 weeks postoperative) parathyroidecto- 
mized animals indicate an increase in the thy- 
roid light cell population relative to normal 
animals. These data, when correlated with re- 
ports of thyrocalcitonin accumulation under 
the- same experimental conditions, support the 
concept that thyroid light cells are the seat of 
thyrocalcitonin production and storage. Cyto- 
logically most of the light cells in the experi- 
mental animals are essentially filled with typ- 
ical secretory vesicles. (Dr. Wetzel and Drs. 
Gittes and Wells [Surg-NCI] ) 

Mucosaccharide Histochemistry 

Investigation of the histochemical charac- 
teristics of epithelial mucosubstances has been 
extended, applying a variety of histochemical 
methods specific for carbohydrate to the study 
of secretions in salivary glands of several spe- 
cies including ungulates, primates, dogs and 
.cats. A number of sulfated mucosubstances 
unfamiliar and unexplored by biochemical 
techniques have been demonstrated and par- 
tially characterized by these methods. In addi- 
tion, various types of sialomucins have been 
identified in separate loci. (Drs. Leppi and 
Spicer) 

The epithelial secretions in the dog's stom- 
ach have been investigated in regard to the 
identification of the types of mucosaccharides 
formed in the different epithelial cells. (Dr. 
Spicer and Dr. Sun [VA Hospital, Phoenix, 
Ariz.] and Dr. Hollander [Mount Sinai Hos- 
pital, New York, N.Y. 



A technique employing dialyzed iron stain- 
ing of cryostat sections has been developed for 
localization of acid mucosubstances at the ul- 
trastructural level and this procedure has been 
applied to investigation of mucosaccharides in 
several sites. (Mrs. Wetzel, Drs. Wetzel and 
Spicer) 

Staining of Paneth cell granules in the 
mouse small intestine has been investigated by 
correlated light microscope and electron mi- 
croscope techniques. Acid mucosubstances in- 
cluding sialo- and sulfomucin have been ob- 
served in the rim of the granules and basic 
protein and an acid mucosubstance in the core 
of the granules. (Dr. Spicer, Mrs. Staley, Mrs. 
Wetzel and Dr. Wetzel) 

Cytochemical studies on the sweat glands 
have been undertaken correlating light micro- 
scope and electron microscope techniques. 
These investigations concern particularly the 
localization of acid mucosubstances and en- 
zymes in the sweat gland epithelium. (Drs. 
Grand and Spicer) 

Correlated light and electron microscope 
studies on the mucosubstance in a coccolitho- 
phorid has revealed sulfate-containing muco- 
saccharides associated with foci of calcium 
deposition in the cytoplasm of this organism 
and in its intercellular matrix. (Dr. Douglas, 
Dr. Isenberg [Long Island Jewish Hospital, 
N.Y.], and Dr. Spicer) 

Immunocytochemical methods employing 
the ferritin-labeled antibody technique at the 
fine structural level reveal selective localiza- 
tion of ferritin-labeled protein in the medial 
A-bands of rat skeletal muscle apparently on 
a nonimmunospecific basis. (Dr. Douglas, Dr. 
Gottlieb [LMP-NIAMD], Dr. Strauss [LI- 
NIAID], and Dr. Spicer) 

Electron microscopy of the thymus of the 
red-eared turtle, Pseudemys scripta elegans, 
has revealed the presence of cells with the sar-